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Part IMolecular Componentsof CellsCHAPTER 1Chemistry Is the Logic of Biological PhenomenaCHAPTER 2Water, pH, and Ionic EquilibriaCHAPTER 3Thermodynamics of Biological SystemsCHAPTER 4Amino AcidsCHAPTER 5Proteins: Their Biological Functionsand Primary StructureAPPENDIX TO CHAPTER 5Protein TechniquesCHAPTER 6Proteins: Secondary, Tertiary, andQuaternary StructureCHAPTER 7CarbohydratesCHAPTER 8LipidsCHAPTER 9Membranes and Cell SurfacesCHAPTER 10Membrane TransportCHAPTER 11NucleotidesCHAPTER 12Nucleic AcidsCHAPTER 13Recombinant DNAAll life depends on water; all organisms are aqueous chemical systems. (Waves in Oahu,Hawaii, Brad Lewis/Liaison International)Chapter 1Chemistry Is the Logic ofBiological PhenomenaOUTLINE1.1 ● Distinctive Properties of Living Systems1.2 ● Biomolecules: The Molecules of Life1.3 ● A Biomolecular Hierarchy: SimpleMolecules Are the Units for BuildingComplex Structures1.4 ● Properties of Biomolecules ReflectTheir Fitness to the Living Condition1.5 ● Organization and Structure of Cells1.6 ● Viruses Are Supramolecular AssembliesActing as Cell Parasites2“ . . . everything that living things do can be understood in terms of the jigglings andwigglings of atoms.”RICHARD P. FEYNMANLectures on PhysicsAddison-Wesley Publishing Company, 1963Molecules are lifeless. Yet, in appropriate complexity and number, moleculescompose living things. These living systems are distinct from the inanimateworld because they have certain extraordinary properties. They can grow, move,perform the incredible chemistry of metabolism, respond to stimuli from theenvironment, and, most significantly, replicate themselves with exceptionalfidelity. The complex structure and behavior of living organisms veil the basictruth that their molecular constitution can be described and understood. Thechemistry of the living cell resembles the chemistry of organic reactions.“Swamp Animals and Birds on the River Gambia,” c. 1912 by Harry Hamilton Johnston (1858–1927). (Royal Geographical Society, London/TheBridgeman Art Library.)Indeed, cellular constituents or biomolecules must conform to the chemicaland physical principles that govern all matter. Despite the spectacular diversityof life, the intricacy of biological structures, and the complexity of vital mech-anisms, life functions are ultimately interpretable in chemical terms. Chemistryis the logic of biological phenomena.1.1 ● Distinctive Properties of Living SystemsThe most obvious quality of living organisms is that they are complicated andhighly organized (Figure 1.1). For example, organisms large enough to be seenwith the naked eye are composed of many cells, typically of many types. Inturn, these cells possess subcellular structures or organelles, which are com-plex assemblies of very large polymeric molecules or macromolecules. Thesemacromolecules themselves show an exquisite degree of organization in theirintricate three-dimensional architecture, even though they are composed ofsimple sets of chemical building blocks, such as sugars and amino acids. Indeed,the complex three-dimensional structure of a macromolecule, known as its conformation, is a consequence of interactions between the monomeric units,according to their individual chemical properties.Biological structures serve functional purposes. That is, biological structureshave a role in terms of the organism’s existence. From parts of organisms, suchas limbs and organs, down to the chemical agents of metabolism, such asenzymes and metabolic intermediates, a biological purpose can be given foreach component. Indeed, it is this functional characteristic of biological struc-tures that separates the science of biology from studies of the inanimate worldsuch as chemistry, physics, and geology. In biology, it is always meaningful toseek the purpose of observed structures, organizations, or patterns, that is, toask what functional role they serve within the organism. Living systems are actively engaged in energy transformations. The maintenanceof the highly organized structure and activity of living systems depends upontheir ability to extract energy from the environment. The ultimate source ofenergy is the sun. Solar energy flows from photosynthetic organisms (those organ-isms able to capture light energy by the process of photosynthesis) throughlogic ● a system of reasoning, using principles of valid inference1.1 ● Distinctive Properties of Living Systems 3FIGURE 1.1 ● (a) Mandrill (Mandrillus sphinx), a baboon native to West Africa. (b) Tropical orchid (Bulbophyllum blumei), New Guinea. (a, Tony Angermayer/Photo Researchers, Inc.;b, Thomas C. Boydon/Marie Selby Botanical Gardens)(a) (b)food chains to herbivores and ultimately to carnivorous predators at the apexof the food pyramid (Figure 1.2). The biosphere is thus a system through whichenergy flows. Organisms capture some of this energy, be it from photosynthe-sis or the metabolism of food, by forming special energized biomolecules, ofwhich ATP and NADPH are the two most prominent examples (Figure 1.3).(Commonly used abbreviations such as ATP and NADPH are defined on theinside back cover of this book.) ATP and NADPH are energized biomoleculesbecause they represent chemically useful forms of stored energy. We explorethe chemical basis of this stored energy in subsequent chapters. For now, suf-fice it to say that when these molecules react with other molecules in the cell,the energy released can be used to drive unfavorable processes. That is, ATP,NADPH, and related compounds are the power sources that drive the energy-requiring activities of the cell, including biosynthesis, movement, osmotic workagainst concentration gradients, and, in special instances, light emission (bio-luminescence). Only upon death does an organism reach equilibrium with itsinanimate environment. The living state is characterized by the flow of energy throughthe organism. At the expense of this energy flow, the organism can maintain its4 Chapter 1 ● Chemistry Is the Logic of Biological PhenomenaFIGURE 1.2 ● The food pyramid. Photosynthetic organisms at the base capture lightenergy. Herbivores and carnivores derive their energy ultimately from these primary pro-ducers.FIGURE 1.3 ● ATP and NADPH, two biochemically important energy-rich compounds.OCH2OH HNH HOH OHOOPO––ONNNNH2ATPH2CONH HCNH2OCH2OOPNADPHOOPO–OPO–OH HH HOH OHOOPO–OPO–OOH HNH HOHNNNNH2O–O–intricate order and activity far removed from equilibrium with its surround-ings, yet exist in a state of apparent constancy over time. This state of appar-ent constancy, or so-called steady-state, is actually a very dynamic condition:energy and material are consumed by the organism and used to maintain itsstability and order. In contrast, inanimate matter, as exemplified by the uni-verse in totality, is moving to a condition of increasing disorder or, in ther-modynamic terms, maximum entropy.Living systems have a remarkable capacity for self-replication. Generation aftergeneration, organisms reproduce virtually identical copies of themselves. Thisself-replication can proceed by a variety of mechanisms, ranging from simpledivision in bacteria to sexual reproduction in plants and animals, but in everycase, it is characterized by an astounding degree of fidelity (Figure 1.4). Indeed,if the accuracy of self-replication were significantly greater, the evolution oforganisms would be hampered. This is so because evolution depends upon nat-ural selectionoperating on individual organisms that vary slightly in their fit-ness for the environment. The fidelity of self-replication resides ultimately inthe chemical nature of the genetic material. This substance consists of poly-meric chains of deoxyribonucleic acid, or DNA, which are structurally com-plementary to one another (Figure 1.5). These molecules can generate newcopies of themselves in a rigorously executed polymerization process thatensures a faithful reproduction of the original DNA strands. In contrast, the1.1 ● Distinctive Properties of Living Systems 5FIGURE 1.4 ● Organisms resemble their par-ents. (a) Reg Garrett with sons Robert, Jeffrey,Randal, and grandson Jackson. (b) Orangutanwith infant. (c) The Grishams: Andrew,Rosemary, Charles, Emily, and David. (a, WilliamW. Garrett, II; b, Randal Harrison Garrett; c, Charles Y. Sipe)(a)(c)(b)molecules of the inanimate world lack this capacity to replicate. A crude mech-anism of replication, or specification of unique chemical structure accordingto some blueprint, must have existed at life’s origin. This primordial system nodoubt shared the property of structural complementarity (see later section)with the highly evolved patterns of replication prevailing today.1.2 ● Biomolecules: The Molecules of LifeThe elemental composition of living matter differs markedly from the relativeabundance of elements in the earth’s crust (Table 1.1). Hydrogen, oxygen, car-bon, and nitrogen constitute more than 99% of the atoms in the human body,with most of the H and O occurring as H2O. Oxygen, silicon, aluminum, andiron are the most abundant atoms in the earth’s crust, with hydrogen, carbon,and nitrogen being relatively rare (less than 0.2% each). Nitrogen as dinitro-gen (N2) is the predominant gas in the atmosphere, and carbon dioxide (CO2)is present at a level of 0.05%, a small but critical amount. Oxygen is also abun-dant in the atmosphere and in the oceans. What property unites H, O, C, and6 Chapter 1 ● Chemistry Is the Logic of Biological PhenomenaTable 1.1Composition of the Earth’s Crust, Seawater, and the Human Body*Earth’s Crust Seawater Human Body†Element % Compound mM Element %O 47 Cl� 548 H 63Si 28 Na� 470 O 25.5Al 7.9 Mg2� 54 C 9.5Fe 4.5 SO42� 28 N 1.4Ca 3.5 Ca2� 10 Ca 0.31Na 2.5 K� 10 P 0.22K 2.5 HCO3� 2.3 Cl 0.08Mg 2.2 NO3� 0.01 K 0.06Ti 0.46 HPO42� �0.001 S 0.05H 0.22 Na 0.03C 0.19 Mg 0.01*Figures for the earth’s crust and the human body are presented as percentages of the totalnumber of atoms; seawater data are millimoles per liter. Figures for the earth’s crust do notinclude water, whereas figures for the human body do.†Trace elements found in the human body serving essential biological functions include Mn,Fe, Co, Cu, Zn, Mo, I, Ni, and Se.complementary ● completing, makingwhole or perfect by combining or filling adeficiencyFIGURE 1.5 ● The DNA double helix. Two complementary polynucleotide chains run-ning in opposite directions can pair through hydrogen bonding between their nitroge-nous bases. Their complementary nucleotide sequences give rise to structural complemen-tarity.AGCAAAAA3'5'5'3'TTTTT TC C CCCG GGG GN and renders these atoms so suitable to the chemistry of life? It is their abil-ity to form covalent bonds by electron-pair sharing. Furthermore, H, C, N, andO are among the lightest elements of the periodic table capable of formingsuch bonds (Figure 1.6). Because the strength of covalent bonds is inverselyproportional to the atomic weights of the atoms involved, H, C, N, and O formthe strongest covalent bonds. Two other covalent bond-forming elements, phos-phorus (as phosphate OOPO32� derivatives) and sulfur, also play importantroles in biomolecules.Biomolecules Are Carbon CompoundsAll biomolecules contain carbon. The prevalence of C is due to its unparal-leled versatility in forming stable covalent bonds by electron-pair sharing.Carbon can form as many as four such bonds by sharing each of the four elec-trons in its outer shell with electrons contributed by other atoms. Atoms com-monly found in covalent linkage to C are C itself, H, O, and N. Hydrogen canform one such bond by contributing its single electron to formation of an elec-tron pair. Oxygen, with two unpaired electrons in its outer shell, can partici-pate in two covalent bonds, and nitrogen, which has three unshared electrons,can form three such covalent bonds. Furthermore, C, N, and O can share twoelectron pairs to form double bonds with one another within biomolecules, aproperty that enhances their chemical versatility. Carbon and nitrogen can evenshare three electron pairs to form triple bonds.1.2 ● Biomolecules: The Molecules of Life 7FIGURE 1.6 ● Covalent bond for-mation by e� pair sharing.H + H H HAtoms e– pairingCovalent bondBond energy(kJ/mol)C + H C+ C+ N N+ O O O+ CC CN NO O+ OO+ N N+ N HH+ O HH414343292351615615686142402946393460HHC CCCC C+ NC+ OCOO+ OONNN HHOCCCCCCCOOONNNOHHC CC NCCCOOO4368 Chapter 1 ● Chemistry Is the Logic of Biological PhenomenaFIGURE 1.7 ● Examples ofthe versatility of COC bonds inbuilding complex structures:linear aliphatic, cyclic,branched, and planar.LINEAR ALIPHATIC:Stearic acidHOOC (CH2)16 CH3 COHOCCH HH HCCH HH HCCH HH HCCH HH HCCH HH HCCH HH HCCH HH HCCH HH HCHH HBRANCHED:β-caroteneH3C CH3CH3CH3 CH3CH3 CH3 H3C CH3H3CCYCLIC:Cholesterol H C CH2CH3HOH3CH3CCH2 CH2 C CH3HCH3PLANAR:Chlorophyll aNNN Mg2+H3C CH2CH3CH3OC OCH3OCH2H3CH3CHCH2CCH2COHCHO CHCCH3HCHHCHHCHCCH3HCHHCHHCHCCH3HCHHCHHCHCCH3HCHHH H HNTwo properties of carbon covalent bonds merit particular attention. Oneis the ability of carbon to form covalent bonds with itself. The other is the tetra-hedral nature of the four covalent bonds when carbon atoms form only singlebonds. Together these properties hold the potential for an incredible varietyof linear, branched, and cyclic compounds of C. This diversity is multiplied fur-ther by the possibilities for including N, O, and H atoms in these compounds(Figure 1.7). We can therefore envision the ability of C to generate complexstructures in three dimensions. These structures, by virtue of appropriatelyincluded N, O, and H atoms, can display unique chemistries suitable to the liv-ing state. Thus, we may ask, is there any pattern or underlying organizationthat brings order to this astounding potentiality?1.3 ● A Biomolecular Hierarchy: Simple Molecules Are theUnits for Building Complex StructuresExamination of the chemical composition of cells reveals a dazzling variety oforganic compounds covering a wide range of molecular dimensions (Table1.2). As this complexity is sorted out and biomolecules are classified accord-ing to the similarities in size and chemical properties, an organizational pat-1.3 ● A Biomolecular Hierarchy: Simple Molecules Are the Units for Building Complex Structures 9Table 1.2Biomolecular DimensionsThe dimensions of mass* and length for biomolecules are given typically in daltons and nanometers,† respectively.One dalton (D) is the mass of one hydrogen atom, 1.67 �10�24 g. One nanometer (nm) is 10�9 m, or 10 Å(angstroms).MassLengthBiomolecule (long dimension, nm) Daltons PicogramsWater 20,0000.3 40,000,018Alanine 20,0000.5 40,000,089Glucose 20,0000.7 40,000,180Phospholipid 20,0003.5 40,000,750Ribonuclease (a small protein) 20,004 40,012,600Immunoglobulin G (IgG) 20,014 40,150,000Myosin (a large muscle protein) 20,160 40,470,000Ribosome (bacteria) 20,018 42,520,000Bacteriophage �X174 (a very small bacterial virus) 20,025 44,700,000Pyruvate dehydrogenase complex (a multienzyme complex) 20,060 47,000,000Tobacco mosaic virus (a plant virus) 20,300 40,000,000 8,006.68 � 10�5Mitochondrion (liver) 21,500 1.5Escherichia coli cell 22,000 2Chloroplast (spinach leaf) 28,000 60Liver cell 20,000 8,000*Molecular mass is expressed in units of daltons (D) or kilodaltons (kD) in this book; alternatively, the dimension-less term molecular weight, symbolized by Mr, and defined as the ratio of the mass of a molecule to 1 dalton of mass, is used.†Prefixes used for powers of 10 are106 mega M 10�3 milli m103 kilo k 10�6 micro �10�1 deci d 10�9 nano n10�2 centi c 10�12 pico p10�15 femto ftern emerges. The molecular constituents of living matter do not reflect ran-domly the infinite possibilities for combining C, H, O, and N atoms. Instead,only a limited set of the many possibilities is found, and these collections sharecertain properties essential to the establishment and maintenance of the liv-ing state. The most prominent aspect of biomolecular organization is thatmacromolecular structures are constructed from simple molecules accordingto a hierarchy of increasing structural complexity. What properties do thesebiomolecules possess that make them so appropriate for the condition of life?Metabolites and MacromoleculesThe major precursors for the formation of biomolecules are water, carbon di-oxide, and three inorganic nitrogen compounds—ammonium (NH4�), nitrate(NO3�), and dinitrogen (N2). Metabolic processes assimilate and transformthese inorganic precursors through ever more complex levels of biomolecularorder (Figure 1.8). In the first step, precursors are converted to metabolites,simple organic compounds that are intermediates in cellular energy transfor-mation and in the biosynthesis of various sets of building blocks: amino acids,sugars, nucleotides, fatty acids, and glycerol. By covalent linkage of these build-ing blocks, the macromolecules are constructed: proteins, polysaccharides,polynucleotides (DNA and RNA), and lipids. (Strictly speaking, lipids containrelatively few building blocks and are therefore not really polymeric like othermacromolecules; however, lipids are important contributors to higher levels ofcomplexity.) Interactions among macromolecules lead to the next level of struc-tural organization, supramolecular complexes. Here, various members of oneor more of the classes of macromolecules come together to form specific assem-blies serving important subcellular functions. Examples of these supramolec-ular assemblies are multifunctional enzyme complexes, ribosomes, chromo-somes, and cytoskeletal elements. For example, a eukaryotic ribosome containsfour different RNA molecules and at least 70 unique proteins. These supramo-lecular assemblies are an interesting contrast to their components because theirstructural integrity is maintained by noncovalent forces, not by covalent bonds.These noncovalent forces include hydrogen bonds, ionic attractions, van derWaals forces, and hydrophobic interactions between macromolecules. Suchforces maintain these supramolecular assemblies in a highly ordered functionalstate. Although noncovalent forces are weak (less than 40 kJ/mol), they arenumerous in these assemblies and thus can collectively maintain the essentialarchitecture of the supramolecular complex under conditions of temperature,pH, and ionic strength that are consistent with cell life.OrganellesThe next higher rung in the hierarchical ladder is occupied by the organelles,entities of considerable dimensions compared to the cell itself. Organelles arefound only in eukaryotic cells, that is, the cells of “higher” organisms (eukary-otic cells are described in Section 1.5). Several kinds, such as mitochondriaand chloroplasts, evolved from bacteria that gained entry to the cytoplasm ofearly eukaryotic cells. Organelles share two attributes: they are cellular inclu-sions, usually membrane bounded, and are dedicated to important cellulartasks. Organelles include the nucleus, mitochondria, chloroplasts, endoplas-mic reticulum, Golgi apparatus, and vacuoles as well as other relatively smallcellular inclusions, such as peroxisomes, lysosomes, and chromoplasts. Thenucleus is the repository of genetic information as contained within the linearsequences of nucleotides in the DNA of chromosomes. Mitochondria are the10 Chapter 1 ● Chemistry Is the Logic of Biological Phenomena“power plants” of cells by virtue of their ability to carry out the energy-releas-ing aerobic metabolism of carbohydrates and fatty acids, capturing the energyin metabolically useful forms such as ATP. Chloroplasts endow cells with theability to carry out photosynthesis. They are the biological agents for harvest-ing light energy and transforming it into metabolically useful chemical forms.11FIGURE 1.8 ● Molecular organization in thecell is a hierarchy.MembranesMembranes define the boundaries of cells and organelles. As such, they arenot easily classified as supramolecular assemblies or organelles, although theyshare the properties of both. Membranes resemble supramolecular complexesin their construction because they are complexes of proteins and lipids main-tained by noncovalent forces. Hydrophobic interactions are particularly impor-tant in maintaining membrane structure. Hydrophobic interactions arisebecause water molecules prefer to interact with each other rather than withnonpolar substances. The presence of nonpolar molecules lessens the rangeof opportunities for water–water interaction by forcing the water moleculesinto ordered arrays around the nonpolar groups. Such ordering can be mini-mized if the individual nonpolar molecules redistribute from a dispersed statein the water into an aggregated organic phase surrounded by water. The spon-taneous assembly of membranes in the aqueous environment where life aroseand exists is the natural result of the hydrophobic (“water-fearing”) characterof their lipids and proteins. Hydrophobic interactions are the creative meansof membrane formation and the driving force that presumably established theboundary of the first cell. The membranes of organelles, such as nuclei, mito-chondria, and chloroplasts, differ from one another, with each having a charac-teristic protein and lipid composition suited to the organelle’s function.Furthermore, the creation of discrete volumes or compartments within cells isnot only an inevitable consequence of the presence of membranes but usuallyan essential condition for proper organellar function.The Unit of Life Is the CellThe cell is characterized as the unit of life, the smallest entity capable of dis-playing the attributes associated uniquely with the living state: growth, metabo-lism, stimulus response, and replication. In the previous discussions, we explic-itly narrowed the infinity of chemical complexity potentially available to organiclife, and we previewed an organizational arrangement, moving from simple tocomplex, that provides interesting insights into the functional and structuralplan of the cell. Nevertheless, we find no obvious explanation within these fea-tures for the living characteristics of cells. Can we find other themes repre-sented within biomolecules that are explicitly chemical yet anticipate or illu-minate the livingcondition?1.4 ● Properties of Biomolecules Reflect Their Fitness to the Living ConditionIf we consider what attributes of biomolecules render them so fit as compo-nents of growing, replicating systems, several biologically relevant themes ofstructure and organization emerge. Furthermore, as we study biochemistry, wewill see that these themes serve as principles of biochemistry. Prominent amongthem is the necessity for information and energy in the maintenance of the living state.Some biomolecules must have the capacity to contain the information or“recipe” of life. Other biomolecules must have the capacity to translate thisinformation so that the blueprint is transformed into the functional, organizedstructures essential to life. Interactions between these structures are theprocesses of life. An orderly mechanism for abstracting energy from the envi-ronment must also exist in order to obtain the energy needed to drive theseprocesses. What properties of biomolecules endow them with the potential forsuch remarkable qualities?12 Chapter 1 ● Chemistry Is the Logic of Biological PhenomenaBiological Macromolecules and Their Building Blocks Have a “Sense” or DirectionalityThe macromolecules of cells are built of units—amino acids in proteins,nucleotides in nucleic acids, and carbohydrates in polysaccharides—that havestructural polarity. That is, these molecules are not symmetrical, and so theycan be thought of as having a “head” and a “tail.” Polymerization of these unitsto form macromolecules occurs by head-to-tail linear connections. Because ofthis, the polymer also has a head and a tail, and hence, the macromoleculehas a “sense” or direction to its structure (Figure 1.9).1.4 ● Properties of Biomolecules Reflect Their Fitness to the Living Condition 13FIGURE 1.9 ● (a) Amino acids build proteins by connecting the �-carboxyl C atom ofone amino acid to the �-amino N atom of the next amino acid in line. (b) Polysaccharidesare built by combining the C-1 of one sugar to the C-4 O of the next sugar in the polymer.(c) Nucleic acids are polymers of nucleotides linked by bonds between the 3�-OH of theribose ring of one nucleotide to the 5�-PO4 of its neighboring nucleotide. All three ofthese polymerization processes involve bond formations accompanied by the eliminationof water (dehydration synthesis reactions). PolysaccharideCOO–+CH3NH R1OAmino acid PolypeptideN CSenseHOCH2OHOHO123456Sugar+4 1SenseHO OHOOCH2PO–NNNH2OH5'4'3' 2'1'+HONucleotidePO–OONucleic acidPO4Sense5' 3'COO–H3NAmino acidCNH R2COO–CH2OHOHO123456SugarHOCH2OHOO1 CH2OHOHO4OOOCH2PO–NNNH2OH5'4'3' 2'1'HONucleotideONNOOCH2PO–N ONNH25'3' 2'HOOOOCH2NNNH2OH3'NNCH R2H3NCCH R1HHOH2OH2OH2OHO HOHOHOHOHOHOOOH OHOH(a)(b)(c)OH..........................................HO+++Biological Macromolecules Are InformationalBecause biological macromolecules have a sense to their structure, the sequen-tial order of their component building blocks, when read along the length ofthe molecule, has the capacity to specify information in the same manner thatthe letters of the alphabet can form words when arranged in a linear sequence(Figure 1.10). Not all biological macromolecules are rich in information.Polysaccharides are often composed of the same sugar unit repeated over andover, as in cellulose or starch, which are homopolymers of many glucose units.On the other hand, proteins and polynucleotides are typically composed ofbuilding blocks arranged in no obvious repetitive way; that is, their sequencesare unique, akin to the letters and punctuation that form this descriptive sen-tence. In these unique sequences lies meaning. To discern the meaning, how-ever, requires some mechanism for recognition.Biomolecules Have Characteristic Three-Dimensional ArchitectureThe structure of any molecule is a unique and specific aspect of its identity.Molecular structure reaches its pinnacle in the intricate complexity of biologi-cal macromolecules, particularly the proteins. Although proteins are linearsequences of covalently linked amino acids, the course of the protein chaincan turn, fold, and coil in the three dimensions of space to establish a specific,highly ordered architecture that is an identifying characteristic of the givenprotein molecule (Figure 1.11).Weak Forces Maintain Biological Structure and DetermineBiomolecular InteractionsCovalent bonds hold atoms together so that molecules are formed. In contrast,weak chemical forces or noncovalent bonds, (hydrogen bonds, van der Waalsforces, ionic interactions, and hydrophobic interactions) are intramolecular orintermolecular attractions between atoms. None of these forces, which typicallyrange from 4 to 30 kJ/mol, are strong enough to bind free atoms together(Table 1.3). The average kinetic energy of molecules at 25°C is 2.5 kJ/mol, sothe energy of weak forces is only several times greater than the dissociatingtendency due to thermal motion of molecules. Thus, these weak forces createinteractions that are constantly forming and breaking at physiological tem-perature, unless by cumulative number they impart stability to the structuresgenerated by their collective action. These weak forces merit further discus-sion because their attributes profoundly influence the nature of the biologicalstructures they build.14 Chapter 1 ● Chemistry Is the Logic of Biological PhenomenaFIGURE 1.10 ● The sequence of monomericunits in a biological polymer has the potentialto contain information if the diversity andorder of the units are not overly simple orrepetitive. Nucleic acids and proteins are infor-mation-rich molecules; polysaccharides are not.FIGURE 1.11 ● Three-dimensional space-filling representation of part of a protein molecule, the antigen-binding domain ofimmunoglobulin G (IgG). Immunoglobulin Gis a major type of circulating antibody. Each ofthe spheres represents an atom in the struc-ture.5' 3'T CA G CA G G T C A G C C A T A G A G T C T AA strand of DNAA polypeptide segmentPhe Ser LysAsn Gly Pro Thr GluA polysaccharide chainGlc Glc Glc Glc Glc Glc Glc Glc GlcVan der Waals Attractive ForcesVan der Waals forces are the result of induced electrical interactions betweenclosely approaching atoms or molecules as their negatively-charged electronclouds fluctuate instantaneously in time. These fluctuations allow attractionsto occur between the positively charged nuclei and the electrons of nearbyatoms. Van der Waals interactions include dipole–dipole interactions, whoseinteraction energies decrease as 1/r3; dipole-induced dipole interactions,which fall off as 1/r5; and induced dipole-induced dipole interactions, oftencalled dispersion or London dispersion forces, which diminish as 1/r6.Dispersion forces contribute to the attractive intermolecular forces between allmolecules, even those without permanent dipoles, and are thus generally moreimportant than dipole–dipole attractions. Van der Waals attractions operateonly over a limited interatomic distance and are an effective bonding interac-tion at physiological temperatures only when a number of atoms in a moleculecan interact with several atoms in a neighboring molecule. For this to occur,the atoms on interacting molecules must pack together neatly. That is, theirmolecular surfaces must possessa degree of structural complementarity (Figure1.12).At best, van der Waals interactions are weak and individually contribute0.4 to 4.0 kJ/mol of stabilization energy. However, the sum of many such inter-actions within a macromolecule or between macromolecules can be substan-tial. For example, model studies of heats of sublimation show that each methy-lene group in a crystalline hydrocarbon accounts for 8 kJ, and each COH groupin a benzene crystal contributes 7 kJ of van der Waals energy per mole.Calculations indicate that the attractive van der Waals energy between theenzyme lysozyme and a sugar substrate that it binds is about 60 kJ/mol.1.4 ● Properties of Biomolecules Reflect Their Fitness to the Living Condition 15Table 1.3Weak Chemical Forces and Their Relative Strengths and DistancesStrength DistanceForce (kJ/mol) (nm) DescriptionVan der Waals interactions 0.4–4.0 0.2 Strength depends on the relative size of the atoms or moleculesand the distance between them. The size factor determines thearea of contact between two molecules: The greater the area,the stronger the interaction.Hydrogen bonds 12–30 0.3 Relative strength is proportional to the polarity of the H bonddonor and H bond acceptor. More polar atoms form strongerH bonds.Ionic interactions 20 0.25 Strength also depends on the relative polarity of the interactingcharged species. Some ionic interactions are also H bonds:ONH3� . . . �OOCOHydrophobic interactions �40 — Force is a complex phenomenon determined by the degree towhich the structure of water is disordered as discretehydrophobic molecules or molecular regions coalesce.FIGURE 1.12 ● Van der Waals packing is enhanced in molecules that are structurallycomplementary. Gln121 represents a surface protuberance on the protein lysozyme. Thisprotuberance fits nicely within a pocket (formed by Tyr101, Tyr32, Phe91, and Trp92) in theantigen-binding domain of an antibody raised against lysozyme. (See also Figure 1.16.) (a) A space-filling representation. (b) A ball-and-stick model. (From Science 233:751 (1986 ), figure 5.) ...(a)(b)Tyr 32Phe 91Trp 92Gln 121Tyr 101When two atoms approach each other so closely that their electron cloudsinterpenetrate, strong repulsion occurs. Such repulsive van der Waals forces fol-low an inverse 12th-power dependence on r (1/r12), as shown in Figure 1.13.Between the repulsive and attractive domains lies a low point in the potentialcurve. This low point defines the distance known as the van der Waals contactdistance, which is the interatomic distance that results if only van der Waalsforces hold two atoms together. The limit of approach of two atoms is deter-mined by the sum of their van der Waals radii (Table 1.4).Hydrogen BondsHydrogen bonds form between a hydrogen atom covalently bonded to an elec-tronegative atom (such as oxygen or nitrogen) and a second electronegativeatom that serves as the hydrogen bond acceptor. Several important biologicalexamples are given in Figure 1.14. Hydrogen bonds, at a strength of 12 to 30kJ/mol, are stronger than van der Waals forces and have an additional prop-erty: H bonds tend to be highly directional, forming straight bonds betweendonor, hydrogen, and acceptor atoms. Hydrogen bonds are also more specificthan van der Waals interactions because they require the presence of comple-mentary hydrogen donor and acceptor groups.Ionic InteractionsIonic interactions are the result of attractive forces between oppositely chargedpolar functions, such as negative carboxyl groups and positive amino groups(Figure 1.15). These electrostatic forces average about 20 kJ/mol in aqueoussolutions. Typically, the electrical charge is radially distributed, and so theseinteractions may lack the directionality of hydrogen bonds or the precise fit ofvan der Waals interactions. Nevertheless, because the opposite charges arerestricted to sterically defined positions, ionic interactions can impart a highdegree of structural specificity.The strength of electrostatic interactions is highly dependent on the natureof the interacting species and the distance, r, between them. Electrostatic inter-actions may involve ions (species possessing discrete charges), permanentdipoles (having a permanent separation of positive and negative charge), andinduced dipoles (having a temporary separation of positive and negative chargeinduced by the environment). Between two ions, the energy falls off as 1/r.The interaction energy between permanent dipoles falls off as 1/r3, whereasthe energy between an ion and an induced dipole falls off as 1/r4.16 Chapter 1 ● Chemistry Is the Logic of Biological PhenomenaFIGURE 1.13 ● The van der Waals interaction energy profile as a function of the dis-tance, r, between the centers of two atoms. The energy was calculated using the empiricalequation U B/r12 � A/r6. (Values for the parameters B 11.5 � 10�6 kJnm12/mol and A 5.96 � 10�3 kJnm6/mol for the interaction between two carbon atoms are fromLevitt, M., 1974, Journal of Molecular Biology 82:393–420.)FIGURE 1.14 ● Some of the biologically important H bonds and functional groups thatserve as H bond donors and acceptors.C OHOO H O–O H NN H ON+ H ON H NBonded atoms0.27 nm0.26 nm0.29 nm0.30 nm0.29 nm0.31 nmApproximatebond length*Lengths given are distances from the atom covalently linked to the H to the atomH-bonded to the hydrogen:O H O0.27 nmFunctional groups which are important Hbond donors and acceptors:COHONHHNHRDonors AcceptorsC OOR ROHNP OO H*0r (nm)–1.0Energy (kJ/mol)01.02.00.2 0.4 0.6 0.8Sum ofvan der Waalsradii1.4 ● Properties of Biomolecules Reflect Their Fitness to the Living Condition 17Table 1.4Radii of the Common Atoms of BiomoleculesAtomVan der Waals Covalent representedAtom radius, nm radius, nm to scaleH 0.1 0.037C 0.17 0.077N 0.15 0.070O 0.14 0.066P 0.19 0.096S 0.185 0.104Half-thicknessof an 0.17 —aromaticringFIGURE 1.15 ● Ionic bonds in biological molecules.Histone-DNA complexes in chromosomesOOO–H2CPO–O OOOH2CPO–OOOH2COOOCH2OO–POOCH2OO OPO–CH2OTACGAT.....................DNA(CH 2) 3NHCH2NNH 2+Histone chain...Magnesium ATP–O P OMg2+O–OP OO–OCH2P OO–O... ... ........OOHHONNNNNH2Intramolecular ionic bonds between oppositelycharged groups on amino acid residues in a proteinNH3+H2CCO–OH2C COO– +H3N...(CH2)4COO–...Hydrophobic InteractionsHydrophobic interactions are due to the strong tendency of water to excludenonpolar groups or molecules (see Chapter 2). Hydrophobic interactions arisenot so much because of any intrinsic affinity of nonpolar substances for oneanother (although van der Waals forces do promote the weak bonding of non-polar substances), but because water molecules prefer the stronger interactionsthat they share with one another, compared to their interaction with nonpo-lar molecules. Hydrogen-bonding interactions between polar water moleculescan be more varied and numerous if nonpolar molecules coalesce to form adistinct organic phase. This phase separation raises the entropy of waterbecause fewer water molecules are arranged in orderly arrays around individ-ual nonpolar molecules. It is these preferential interactions between water mol-ecules that “exclude” hydrophobic substances from aqueous solutionand drivethe tendency of nonpolar molecules to cluster together. Thus, nonpolar regionsof biological macromolecules are often buried in the molecule’s interior toexclude them from the aqueous milieu. The formation of oil droplets ashydrophobic nonpolar lipid molecules coalesce in the presence of water is anapproximation of this phenomenon. These tendencies have important conse-quences in the creation and maintenance of the macromolecular structuresand supramolecular assemblies of living cells.Structural Complementarity Determines Biomolecular InteractionsStructural complementarity is the means of recognition in biomolecular inter-actions. The complicated and highly organized patterns of life depend uponthe ability of biomolecules to recognize and interact with one another in veryspecific ways. Such interactions are fundamental to metabolism, growth, repli-cation, and other vital processes. The interaction of one molecule with another,a protein with a metabolite, for example, can be most precise if the structureof one is complementary to the structure of the other, as in two connectingpieces of a puzzle or, in the more popular analogy for macromolecules andtheir ligands, a lock and its key (Figure 1.16). This principle of structural com-plementarity is the very essence of biomolecular recognition. Structural com-plementarity is the significant clue to understanding the functional properties of biologi-cal systems. Biological systems from the macromolecular level to the cellularlevel operate via specific molecular recognition mechanisms based on struc-tural complementarity: a protein recognizes its specific metabolite, a strand ofDNA recognizes its complementary strand, sperm recognize an egg. All theseinteractions involve structural complementarity between molecules.Biomolecular Recognition Is Mediated by Weak Chemical ForcesThe biomolecular recognition events that occur through structural comple-mentarity are mediated by the weak chemical forces previously discussed. It isimportant to realize that, because these interactions are sufficiently weak, theyare readily reversible. Consequently, biomolecular interactions tend to be tran-sient; rigid, static lattices of biomolecules that might paralyze cellular activitiesare not formed. Instead, a dynamic interplay occurs between metabolites andmacromolecules, hormones and receptors, and all the other participants instru-mental to life processes. This interplay is initiated upon specific recognitionbetween complementary molecules and ultimately culminates in unique physi-ological activities. Biological function is achieved through mechanisms based on struc-tural complementarity and weak chemical interactions.18 Chapter 1 ● Chemistry Is the Logic of Biological Phenomenamilieu ● the environment or surroundings;from the French mi meaning “middle” andlieu meaning “place”ligand ● something that binds; a moleculethat is bound to another molecule; from theLatin ligare, meaning “to bind”This principle of structural complementarity extends to higher interactionsessential to the establishment of the living condition. For example, the for-mation of supramolecular complexes occurs because of recognition and inter-action between their various macromolecular components, as governed by theweak forces formed between them. If a sufficient number of weak bonds canbe formed, as in macromolecules complementary in structure to one another,larger structures assemble spontaneously. The tendency for nonpolar mole-cules and parts of molecules to come together through hydrophobic interac-tions also promotes the formation of supramolecular assemblies. Very complexsubcellular structures are actually spontaneously formed in an assembly processthat is driven by weak forces accumulated through structural complementarity.Weak Forces Restrict Organisms to a Narrow Range of Environmental ConditionsThe central role of weak forces in biomolecular interactions restricts living sys-tems to a narrow range of physical conditions. Biological macromolecules arefunctionally active only within a narrow range of environmental conditions,such as temperature, ionic strength, and relative acidity. Extremes of these con-ditions disrupt the weak forces essential to maintaining the intricate structureof macromolecules. The loss of structural order in these complex macromole-cules, so-called denaturation, is accompanied by loss of function (Figure 1.17).As a consequence, cells cannot tolerate reactions in which large amounts ofenergy are released. Nor can they generate a large energy burst to drive energy-requiring processes. Instead, such transformations take place via sequentialseries of chemical reactions whose overall effect achieves dramatic energychanges, even though any given reaction in the series proceeds with only mod-1.4 ● Properties of Biomolecules Reflect Their Fitness to the Living Condition 19FIGURE 1.16 ● Structural complementarity: the pieces of a puzzle, the lock and its key,a biological macromolecule and its ligand—an antigen–antibody complex. (a) The anti-gen on the right (green) is a small protein, lysozyme, from hen egg white. The part of theantibody molecule (IgG) shown on the left in blue and yellow includes the antigen-bindingdomain. (b) This domain has a pocket that is structurally complementary to a surface pro-tuberance (Gln121, shown in red between antigen and antigen-binding domain) on theantigen. (See also Figure 1.12.) (photos, courtesy of Professor Simon E. V. Philips)PuzzleMACRO-MOLEC-ULELock and keyLigandLigandMacromolecule(a)(b)20 Chapter 1 ● Chemistry Is the Logic of Biological PhenomenaFIGURE 1.17 ● Denaturation and renaturationof the intricate structure of a protein.FIGURE 1.18 ● Metabolism is the organized release or capture of small amounts ofenergy in processes whose overall change in energy is large. (a) For example, the combus-tion of glucose by cells is a major pathway of energy production, with the energy capturedappearing as 30 to 38 equivalents of ATP, the principal energy-rich chemical of cells. Theten reactions of glycolysis, the nine reactions of the citric acid cycle, and the successivelinked reactions of oxidative phosphorylation release the energy of glucose in a stepwisefashion and the small “packets” of energy appear in ATP. (b) Combustion of glucose in abomb calorimeter results in an uncontrolled, explosive release of energy in its least usefulform, heat.The combustion of glucose: C6H12O6 + 6O2 6CO2 + 6H2O + 2,870 kJ energy (a) In an aerobic cellGlucose2 Pyruvate6CO2 + 6H2OCitric acid cycleand oxidativephosphorylationGlycolysis30–38 ATP(b) In a bomb calorimeterGlucose2,870 kJenergyas heat6CO2 + 6H2OATPATPATPATPATPATPATPATPATPATPATPATP ATPATPATPATP ATP ATPATPATPest input or release of energy (Figure 1.18). These sequences of reactions areorganized to provide for the release of useful energy to the cell from the break-down of food or to take such energy and use it to drive the synthesis of bio-molecules essential to the living state. Collectively, these reaction sequencesconstitute cellular metabolism—the ordered reaction pathways by which cel-lular chemistry proceeds and biological energy transformations are accom-plished.EnzymesThe sensitivity of cellular constituents to environmental extremes placesanother constraint on the reactions of metabolism. The rate at which cellularreactions proceed is a very important factor in maintenance of the living state.However, the common ways chemists accelerate reactions are not available tocells; the temperature cannot be raised, acid or base cannot be added, the pres-sure cannot be elevated, and concentrations cannotbe dramatically increased.Instead, biomolecular catalysts mediate cellular reactions. These catalysts,called enzymes, accelerate the reaction rates many orders of magnitude and,by selecting the substances undergoing reaction, determine the specific reac-tion taking place. Virtually every metabolic reaction is served by an enzymewhose sole biological purpose is to catalyze its specific reaction (Figure 1.19).Metabolic Regulation Is Achieved by Controlling the Activity of EnzymesThousands of reactions mediated by an equal number of enzymes are occur-ring at any given instant within the cell. Metabolism has many branch points,cycles, and interconnections, as a glance at a metabolic pathway map reveals1.4 ● Properties of Biomolecules Reflect Their Fitness to the Living Condition 21FIGURE 1.19 ● Carbonic anhydrase, a representative enzyme, and the reaction that itcatalyzes. Dissolved carbon dioxide is slowly hydrated by water to form bicarbonate ionand H�:CO2 � H2O 34 HCO3� � H�At 20°C, the rate constant for this uncatalyzed reaction, kuncat, is 0.03/sec. In the presenceof the enzyme carbonic anhydrase, the rate constant for this reaction, kcat, is 106/sec.Thus carbonic anhydrase accelerates the rate of this reaction 3.3 � 107 times. Carbonicanhydrase is a 29-kD protein.22(Figure 1.20). All of these reactions, many of which are at apparent cross-purposes in the cell, must be fine-tuned and integrated so that metabolism andlife proceed harmoniously. The need for metabolic regulation is obvious. Thismetabolic regulation is achieved through controls on enzyme activity so thatthe rates of cellular reactions are appropriate to cellular requirements.Despite the organized pattern of metabolism and the thousands of enzymesrequired, cellular reactions nevertheless conform to the same thermodynamicprinciples that govern any chemical reaction. Enzymes have no influence overenergy changes (the thermodynamic component) in their reactions. Enzymesonly influence reaction rates. Thus, cells are systems that take in food, releasewaste, and carry out complex degradative and biosynthetic reactions essentialto their survival while operating under conditions of essentially constant tem-perature and pressure and maintaining a constant internal environment (homeostasis) with no outwardly apparent changes. Cells are open thermodynamicsystems exchanging matter and energy with their environment and functioning as highlyregulated isothermal chemical engines.1.5 ● Organization and Structure of CellsAll living cells fall into one of two broad categories—prokaryotic and eukary-otic. The distinction is based on whether or not the cell has a nucleus.Prokaryotes are single-celled organisms that lack nuclei and other organelles;the word is derived from pro meaning “prior to” and karyote meaning “nucleus.”In conventional biological classification schemes, prokaryotes are groupedtogether as members of the kingdom Monera, represented by bacteria andcyanobacteria (formerly called blue-green algae). The other four living king-doms are all eukaryotes—the single-celled Protists, such as amoebae, and allmulticellular life forms, including the Fungi, Plant, and Animal kingdoms.Eukaryotic cells have true nuclei and other organelles such as mitochondria,with the prefix eu meaning “true.”Early Evolution of CellsUntil recently, most biologists accepted the idea that eukaryotes evolved fromthe simpler prokaryotes in some linear progression from simple to complexover the course of geological time. Contemporary evidence favors the view thatpresent-day organisms are better grouped into three classes or lineages: eukary-otes and two prokaryotic groups, the eubacteria and the archaea (formerly des-ignated as archaebacteria). All are believed to have evolved approximately 3.5billion years ago from a common ancestral form called the progenote. It is nowunderstood that eukaryotic cells are, in reality, composite cells derived fromvarious prokaryotic contributions. Thus, the dichotomy between prokaryoticcells and eukaryotic cells, although convenient, is an artificial distinction.Despite the great diversity in form and function, cells and organisms sharea common biochemistry. This commonality, although long established, hasreceived further validation through whole genome sequencing, or the deter-mination of the complete nucleotide sequence within the DNA of an organ-ism. For example, the recently sequenced genome of the archaeon Methanococcus1.5 ● Organization and Structure of Cells 23FIGURE 1.20 ● Reproduction of a metabolic map. (Courtesy of D. E. Nicholson, University of Leedsand Sigma Chemical Co., St. Louis, MO.)▲jannaschii shows 44% similarity to known genes in eubacteria and eukaryotes,yet 56% of its genes are new to science. Whole genome sequencing is revolu-tionizing biochemistry as the protein-coding sequences of newly revealed genesoutpace our understanding of what the proteins are and what they do.Structural Organization of Prokaryotic CellsAmong prokaryotes (the simplest cells), most known species are eubacteria andthey form a widely spread group. Certain of them are pathogenic to humans.The archaea are remarkable because they can be found in unusual environ-ments where other cells cannot survive. Archaea include the thermoacidophiles(heat- and acid-loving bacteria) of hot springs, the halophiles (salt-loving bac-teria) of salt lakes and ponds, and the methanogens (bacteria that generatemethane from CO2 and H2). Prokaryotes are typically very small, on the orderof several microns in length, and are usually surrounded by a rigid cell wallthat protects the cell and gives it its shape. The characteristic structural orga-nization of a prokaryotic cell is depicted in Figure 1.21.Prokaryotic cells have only a single membrane, the plasma membrane orcell membrane. Because they have no other membranes, prokaryotic cells con-tain no nucleus or organelles. Nevertheless, they possess a distinct nuclear areawhere a single circular chromosome is localized, and some have an internalmembranous structure called a mesosome that is derived from and continu-ous with the cell membrane. Reactions of cellular respiration are localized onthese membranes. In photosynthetic prokaryotes such as the cyanobacteria,24 Chapter 1 ● Chemistry Is the Logic of Biological PhenomenaFIGURE 1.21 ● This bacterium is Escherichia coli, a member of the coliform group of bacteria that colo-nize the intestinal tract of humans. E. coli organisms have rather simple nutritional requirements. They growand multiply quite well if provided with a simple carbohydrate source of energy (such as glucose), ammo-nium ions as a source of nitrogen, and a few mineral salts. The simple nutrition of this “lower” organismmeans that its biosynthetic capacities must be quite advanced. When growing at 37°C on a rich organicmedium, E. coli cells divide every 20 minutes. Subcellular features include the cell wall, plasma membrane,nuclear region, ribosomes, storage granules, and cytosol (Table 1.5). (photo, Martin Rotker/Phototake, Inc.; inset photo,David M. Phillips/The Population Council/Science Source/Photo Researchers, Inc.)flat, sheetlike membranous structures called lamellae are formed from cellmembrane infoldings. These lamellae are the sites of photosynthetic activity,but in prokaryotes, they are not contained within plastids, the organelles ofphotosynthesis found in higher plant cells. Prokaryotic cells also lack acytoskeleton; the cell wall maintains their structure. Some bacteria have fla-gella, single, long filaments used for motility. Prokaryotes largely reproduce byasexual division, although sexual exchanges can occur. Table 1.5 lists the majorfeatures of prokaryotic cells.Structural Organization of Eukaryotic CellsIn comparison to prokaryotic cells, eukaryotic cells are much greater in size,typically having cell volumes 103 to 104 times larger. Also, they are much morecomplex. These two features require that eukaryotic cells partition their diverse1.5 ● Organization and Structure of Cells 25Table 1.5Major Features of Prokaryotic CellsStructure Molecular Composition FunctionCell wall Peptidoglycan: a rigid framework of Mechanical support, shape, and protectionpolysaccharide cross-linked by short peptide against swelling in hypotonic media. Thechains. Some bacteria possess a cell wall is a porous nonselective barrier thatlipopolysaccharide- and protein-rich outer allows most small molecules to pass.membrane.Cell membrane The cell membrane is composed of about The cell membrane is a highly selective45% lipid and 55% protein. The lipids permeability barrier that controls the entryform a bilayer that is a continuous of most substances into the cell. Importantnonpolar hydrophobic phase in which the enzymes in the generation of cellularproteins are embedded. energy are located in the membrane.Nuclear area or nucleoid The genetic material is a single tightly coiled DNA is the blueprint of the cell, the repositoryDNA molecule 2 nm in diameter but over of the cell’s genetic information. During cell1 mm in length (molecular mass of E. coli division, each strand of the double-strandedDNA is 3 � 109 daltons; 4.64 � 106 DNA molecule is replicated to yield twonucleotide pairs). double-helical daughter molecules.Messenger RNA (mRNA) is transcribed fromDNA to direct the synthesis of cellularproteins.Ribosomes Bacterial cells contain about 15,000 Ribosomes are the sites of protein synthesis.ribosomes. Each is composed of a small The mRNA binds to ribosomes, and the(30S) subunit and a large (50S) subunit. mRNA nucleotide sequence specifies theThe mass of a single ribosome is protein that is synthesized.2.3 � 106 daltons. It consists of 65% RNAand 35% protein.Storage granules Bacteria contain granules that represent When needed as metabolic fuel, the monomericstorage forms of polymerized metabolites units of the polymer are liberated andsuch as sugars or -hydroxybutyric acid. degraded by energy-yielding pathways inthe cell.Cytosol Despite its amorphous appearance, the The cytosol is the site of intermediarycytosol is now recognized to be an metabolism, the interconnecting sets oforganized gelatinous compartment that chemical reactions by which cells generateis 20% protein by weight and rich in energy and form the precursors necessary forthe organic molecules that are the biosynthesis of macromolecules essential tointermediates in metabolism. cell growth and function.metabolic processes into organized compartments, with each compartmentdedicated to a particular function. A system of internal membranes accom-plishes this partitioning. A typical animal cell is shown in Figure 1.22; a typicalplant cell in Figure 1.23. Tables 1.6 and 1.7 list the major features of a typicalanimal cell and a higher plant cell, respectively.Eukaryotic cells possess a discrete, membrane-bounded nucleus, the repos-itory of the cell’s genetic material, which is distributed among a few or manychromosomes. During cell division, equivalent copies of this genetic materialmust be passed to both daughter cells through duplication and orderly parti-tioning of the chromosomes by the process known as mitosis. Like prokaryotic26 Chapter 1 ● Chemistry Is the Logic of Biological PhenomenaFIGURE 1.22 ● This figure diagrams a rat liver cell, a typical higher animal cell in whichthe characteristic features of animal cells are evident, such as a nucleus, nucleolus, mito-chondria, Golgi bodies, lysosomes, and endoplasmic reticulum (ER). Microtubules and thenetwork of filaments constituting the cytoskeleton are also depicted. (photos, top, Dwight R.Kuhn/Visuals Unlimited; middle, D.W. Fawcett/Visuals Unlimited; bottom, Keith Porter/Photo Researchers, Inc.)AN ANIMAL CELLRough endoplasmic reticulum (plant and animal)Smooth endoplasmic reticulum (plant and animal)Mitochondrion (plant and animal)Smooth endoplasmic reticulumNuclear membraneNucleolusNucleusPlasmamembraneGolgi bodyFilamentous cytoskeleton(microtubules)CytoplasmMitochondrionLysosomeRough endoplasmic reticulumTable 1.6Major Features of a Typical Animal CellStructure Molecular Composition FunctionExtracellular matrix The surfaces of animal cells are covered with This complex coating is cell-specific, serves ina flexible and sticky layer of complex cell – cell recognition and communication,carbohydrates, proteins, and lipids. creates cell adhesion, and provides aprotective outer layer.Cell membrane Roughly 50�50 lipid�protein as a 5-nm-thick The plasma membrane is a selectively(plasma membrane) continuous sheet of lipid bilayer in which a permeable outer boundary of the cell,variety of proteins are embedded. containing specific systems—pumps, channels,transporters—for the exchange of nutrientsand other materials with the environment.Important enzymes are also located here.Nucleus The nucleus is separated from the cytosol by The nucleus is the repository of genetica double membrane, the nuclear envelope. information encoded in DNA and organizedThe DNA is complexed with basic proteins into chromosomes. During mitosis, the(histones) to form chromatin fibers, the chromosomes are replicated and transmittedmaterial from which chromosomes are to the daughter cells. The genetic informationmade. A distinct RNA-rich region, the of DNA is transcribed into RNA in thenucleolus, is the site of ribosome assembly. nucleus and passes into the cytosol whereit is translated into protein by ribosomes.Mitochondria Mitochondria are organelles surrounded by Mitochondria are the power plants oftwo membranes that differ markedly in their eukaryotic cells where carbohydrates,protein and lipid composition. The inner fats, and amino acids are oxidized tomembrane and its interior volume, the CO2 and H2O. The energy releasedmatrix, contain many important enzymes of is trapped as high-energy phosphateenergy metabolism. Mitochondria are about bonds in ATP.the size of bacteria, �1 �m. Cells containhundreds of mitochondria, which collectivelyoccupy about one-fifth of the cell volume.Golgi apparatus A system of flattened membrane-bounded Involved in the packaging and processing ofvesicles often stacked into a complex. macromolecules for secretion and forNumerous small vesicles are found delivery to other cellular compartments.peripheral to the Golgi and containsecretory material packaged by the Golgi.Endoplasmic reticulum Flattened sacs, tubes, and sheets of internal The endoplasmic reticulum is a labyrinthine(ER) and ribosomes membrane extending throughout the organelle where both membrane proteinscytoplasm of the cell and enclosing a large and lipids are synthesized. Proteins madeinterconnecting series of volumes called by the ribosomes of the rough ER passcisternae. The ER membrane is continuous through the outer ER membrane into thewith the outer membrane of the nuclear cisternae and can be transported via theenvelope. Portions of the sheetlike areas of Golgi to the periphery of the cell. Otherthe ER are studded with ribosomes, giving ribosomes unassociated with the ER carryrise to rough ER. Eukaryotic ribosomes are on protein synthesis in the cytosol.larger than prokaryotic ribosomes.Lysosomes Lysosomes are vesicles 0.2–0.5 �m in diameter, Lysosomes function in intracellular digestionbounded by a single membrane. They contain of materials entering the cell viahydrolytic enzymes such as proteases and phagocytosis or pinocytosis. They alsonucleases which, if set free, could degrade function in the controlleddegradationessential cell constituents. They are of cellular components.formed by budding from the Golgiapparatus.Peroxisomes Like lysosomes, peroxisomes are 0.2–0.5 �m Peroxisomes act to oxidize certain nutrients,single-membrane–bounded vesicles. They such as amino acids. In doing so, they formcontain a variety of oxidative enzymes that potentially toxic hydrogen peroxide, H2O2,use molecular oxygen and generate peroxides. and then decompose it to H2O and O2 byThey are formed by budding from the smooth way of the peroxide-cleaving enzymeER. catalase.Cytoskeleton The cytoskeleton is composed of a network The cytoskeleton determines the shape of theof protein filaments: actin filaments cell and gives it its ability to move.(or microfilaments), 7 nm in diameter; It also mediates the internal movementsintermediate filaments, 8–10 nm; and that occur in the cytoplasm, such as themicrotubules, 25 nm. These filaments interact migration of organelles and mitoticin establishing the structure and functions movements of chromosomes. The propulsionof the cytoskeleton. This interacting network instruments of cells—cilia and flagella—areof protein filaments gives structure and constructed of microtubules.organization to the cytoplasm.cells, eukaryotic cells are surrounded by a plasma membrane. Unlike prokary-otic cells, eukaryotic cells are rich in internal membranes that are differenti-ated into specialized structures such as the endoplasmic reticulum (ER) andthe Golgi apparatus. Membranes also surround certain organelles (mitochon-dria and chloroplasts, for example) and various vesicles, including vacuoles,lysosomes, and peroxisomes. The common purpose of these membranous par-titionings is the creation of cellular compartments that have specific, organizedmetabolic functions, such as the mitochondrion’s role as the principal site ofcellular energy production. Eukaryotic cells also have a cytoskeleton composedof arrays of filaments that give the cell its shape and its capacity to move. Someeukaryotic cells also have long projections on their surface—cilia or flagella—which provide propulsion.FIGURE 1.23 ● This figure diagrams a cell in the leaf of a higher plant. The cell wall,membrane, nucleus, chloroplasts, mitochondria, vacuole, ER, and other characteristic fea-tures are shown. (photos, top, middle, Dr. Dennis Kunkel/Phototake, NYC; bottom, Biophoto Associates)MitochondrionLysosomeSmooth endoplasmicreticulumNuclearmembraneNucleolusNucleusRough endoplasmicreticulumGolgi bodyPlasma membraneCellulose wallPectinCell wallChloroplastVacuoleChloroplast (plant cell only)A PLANT CELLGolgi body (plant and animal)Nucleus (plant and animal)28 Chapter 1 ● Chemistry Is the Logic of Biological Phenomena1.5 ● Organization and Structure of Cells 29Table 1.7Major Features of a Higher Plant Cell: A Photosynthetic Leaf CellStructure Molecular Composition FunctionCell wall Cellulose fibers embedded in a Protection against osmotic or mechanicalpolysaccharide/protein matrix; it is rupture. The walls of neighboring cellsthick (�0.1 �m), rigid, and porous interact in cementing the cells togetherto small molecules. to form the plant. Channels for fluid circulation and for cell–cellcommunication pass through the walls.The structural material confers formand strength on plant tissue.Cell membrane Plant cell membranes are similar in The plasma membrane of plant cells isoverall structure and organization to selectively permeable, containing transportanimal cell membranes but differ systems for the uptake of essentialin lipid and protein composition. nutrients and inorganic ions. A numberof important enzymes are localized here.Nucleus The nucleus, nucleolus, and nuclear Chromosomal organization, DNA replication,envelope of plant cells are like transcription, ribosome synthesis, andthose of animal cells. mitosis in plant cells are grossly similar tothe analogous features in animals.Chloroplasts Plant cells contain a unique family Chloroplasts are the site of photosynthesis,of organelles, the plastids, of which the reactions by which light energy isthe chloroplast is the prominent converted to metabolically useful chemicalexample. Chloroplasts have a double energy in the form of ATP. These reactionsmembrane envelope, an inner occur on the thylakoid membranes. Thevolume called the stroma, and an formation of carbohydrate from CO2 takesinternal membrane system rich in place in the stroma. Oxygen is evolvedthylakoid membranes, which enclose during photosynthesis. Chloroplasts area third compartment, the thylakoid the primary source of energy in the light.lumen. Chloroplasts are significantlylarger than mitochondria. Other plastidsare found in specialized structuressuch as fruits, flower petals, and rootsand have specialized roles.Mitochondria Plant cell mitochondria resemble the Plant mitochondria are the main source ofmitochondria of other eukaryotes energy generation in photosyntheticin form and function. cells in the dark and in nonphotosyntheticcells under all conditions.Vacuole The vacuole is usually the most Vacuoles function in transport and storageobvious compartment in plant cells. of nutrients and cellular waste products.It is a very large vesicle enclosed By accumulating water, the vacuoleby a single membrane called the allows the plant cell to grow dramaticallytonoplast. Vacuoles tend to be in size with no increase insmaller in young cells, but in mature cytoplasmic volume.cells, they may occupy more than50% of the cell’s volume. Vacuolesoccupy the center of the cell, withthe cytoplasm being locatedperipherally around it. They resemblethe lysosomes of animal cells.Golgi apparatus, endoplasmic Plant cells also contain all of these These organelles serve the same purposesreticulum, ribosomes, characteristic eukaryotic organelles, in plant cells that they do in animallysosomes, peroxisomes, and essentially in the form described for cells.cytoskeleton animal cells.1.6 ● Viruses Are Supramolecular Assemblies Acting as Cell ParasitesViruses are supramolecular complexes of nucleic acid, either DNA or RNA,encapsulated in a protein coat and, in some instances, surrounded by a mem-brane envelope (Figure 1.24). The bits of nucleic acid in viruses are, in real-ity, mobile elements of genetic information. The protein coat serves to protectthe nucleic acid and allows it to gain entry to the cells that are its specific hosts.Viruses unique for all types of cells are known. Viruses infecting bacteria arecalled bacteriophages (“bacteria eaters”); different viruses infect animal cellsand plant cells. Once the nucleic acid of a virus gains access to its specific host,it typically takes over the metabolic machinery of the host cell, diverting it tothe production of virus particles. The host metabolic functions are subjugatedto the synthesis of viral nucleic acid and proteins. Mature virus particles ariseby encapsulating the nucleic acid within a protein coat called the capsid.Viruses are thus supramolecular assemblies that act as parasites of cells (Figure1.25).30 Chapter 1 ● Chemistry Is the Logic of Biological PhenomenaFIGURE 1.24 ● Viruses are genetic elements enclosed in a protein coat. Viruses are notfree-living and can only reproduce within cells. Viruses show an almost absolute specificityfor their particular host cells, infecting and multiplying only within those cells. Viruses areknown for virtually every kind of cell. Shown here are examples of (a) a bacterial virus,bacteriophage T4; (b) an animal virus, adenovirus (inset at greater magnification); and (c) a plant virus, tobacco mosaic virus. (a, M. Wurtz/Biozeentrum/University of Basel/SPL/Photo Researchers, Inc.; b, Dr. Thomas Broker/Phototake,NYC; inset, CNRI/SPL/Photo Researchers, Inc.; c, BiologyMedia/Photo Researchers, Inc.)(c)(a) (b)Often, viruses cause the lysis of the cells they infect. It is their cytolyticproperties that are the basis of viral disease. In certain circumstances, the viralgenetic elements may integrate into the host chromosome and become quies-cent. Such a state is termed lysogeny. Typically, damage to the host cell acti-vates the replicative capacities of the quiescent viral nucleic acid, leading toviral propagation and release. Some viruses are implicated in transforming cellsinto a cancerous state, that is, in converting their hosts to an unregulated stateof cell division and proliferation. Because all viruses are heavily dependent ontheir host for the production of viral progeny, viruses must have arisen aftercells were established in the course of evolution. Presumably, the first viruseswere fragments of nucleic acid that developed the ability to replicate inde-pendently of the chromosome and then acquired the necessary genes enablingprotection, autonomy, and transfer between cells.1.6 ● Viruses Are Supramolecular Assemblies Acting as Cell Parasites 31FIGURE 1.25 ● The virus life cycle. Virusesare mobile bits of genetic information encapsu-lated in a protein coat. The genetic materialcan be either DNA or RNA. Once this geneticmaterial gains entry to its host cell, it takesover the host machinery for macromolecularsynthesis and subverts it to the synthesis ofviral-specific nucleic acids and proteins. Thesevirus components are then assembled intomature virus particles that are released fromthe cell. Often, this parasitic cycle of virusinfection leads to cell death and disease.Protein coatGeneticmaterial(DNA or RNA)Entry of virusgenome into cellTranscriptionRNATranslationAssemblyRelease from cellReplicationHost cellCoat proteinsPROBLEMS1. The nutritional requirements of Escherichia coli cells are farsimpler than those of humans, yet the macromolecules found inbacteria are about as complex as those of animals. Since bacteriacan make all their essential biomolecules while subsisting on a sim-pler diet, do you think bacteria may have more biosynthetic capac-ity and hence more metabolic complexity than animals? Organizeyour thoughts on this question, pro and con, into a rational argu-ment.2. Without consulting chapter figures, sketch the characteristicprokaryotic and eukaryotic cell types and label their pertinentorganelle and membrane systems.3. Escherichia coli cells are about 2 �m (microns) long and 0.8�m in diameter.a. How many E. coli cells laid end to end would fit across thediameter of a pin head? (Assume a pinhead diameter of 0.5mm.)b. What is the volume of an E. coli cell? (Assume it is a cylinder,with the volume of a cylinder given by V � r2h, where � 3.14.)c. What is the surface area of an E. coli cell? What is the surface-to-volume ratio of an E. coli cell?d. Glucose, a major energy-yielding nutrient, is present in bac-terial cells at a concentration of about 1 mM. How many glucosemolecules are contained in a typical E. coli cell? (Recall thatAvogadro’s number 6.023 � 1023.)e. A number of regulatory proteins are present in E. coli at onlyone or two molecules per cell. If we assume that an E. coli cell con-tains just one molecule of a particular protein, what is the molarconcentration of this protein in the cell?f. An E. coli cell contains about 15,000 ribosomes, which carryout protein synthesis. Assuming ribosomes are spherical and havea diameter of 20 nm (nanometers), what fraction of the E. coli cellvolume is occupied by ribosomes?g. The E. coli chromosome is a single DNA molecule whose massis about 3 � 109 daltons. This macromolecule is actually a lineararray of nucleotide pairs. The average molecular weight of anucleotide pair is 660, and each pair imparts 0.34 nm to the lengthof the DNA molecule. What is the total length of the E. coli chro-mosome? How does this length compare with the overall dimen-sions of an E. coli cell? How many nucleotide pairs does this DNAcontain? The average E. coli protein is a linear chain of 360 aminoacids. If three nucleotide pairs in a gene encode one amino acidin a protein, how many different proteins can the E. coli chromo-some encode? (The answer to this question is a reasonable approx-imation of the maximum number of different kinds of proteinsthat can be expected in bacteria.)4. Assume that mitochondria are cylinders 1.5 �m in length and0.6 �m in diameter.a. What is the volume of a single mitochondrion?b. Oxaloacetate is an intermediate in the citric acid cycle, an important metabolic pathway localized in the mitochondria ofeukaryotic cells. The concentration of oxaloacetate in mitochon-dria is about 0.03 �M. How many molecules of oxaloacetate arein a single mitochondrion?5. Assume that liver cells are cuboidal in shape, 20 �m on a side.a. How many liver cells laid end to end would fit across the diam-eter of a pin head? (Assume a pinhead diameter of 0.5 mm.)b. What is the volume of a liver cell? (Assume it is a cube.)c. What is the surface area of a liver cell? What is the surface-to-volume ratio of a liver cell? How does this compare to the surface-to-volume ratio of an E. coli cell (compare this answer to that ofproblem 3c)? What problems must cells with low surface-to-volume ratios confront that do not occur in cells with high surface-to-volume ratios?d. A human liver cell contains two sets of 23 chromosomes, eachset being roughly equivalent in information content. The totalmass of DNA contained in these 46 enormous DNA molecules is4 � 1012 daltons. Since each nucleotide pair contributes 660 dal-tons to the mass of DNA and 0.34 nm to the length of DNA, whatis the total number of nucleotide pairs and the complete lengthof the DNA in a liver cell? How does this length compare with theoverall dimensions of a liver cell? The maximal information ineach set of liver cell chromosomes should be related to the num-ber of nucleotide pairs in the chromosome set’s DNA. This num-ber can be obtained by dividing the total number of nucleotidepairs calculated above by 2. What is this value? If this informationis expressed in proteins that average 400 amino acids in lengthand three nucleotide pairs encode one amino acid in a protein,how many different kinds of proteins might a liver cell be able toproduce? (In reality, liver cells express at most about 30,000 dif-ferent proteins. Thus, a large discrepancy exists between the the-oretical information content of DNA in liver cells and the amountof information actually expressed.)6. Biomolecules interact with one another through molecular sur-faces that are structurally complementary. How can various proteinsinteract with molecules as different as simple ions, hydrophobiclipids, polar but uncharged carbohydrates, and even nucleic acids?7. What structural features allow biological polymers to be infor-mational macromolecules? Is it possible for polysaccharides to beinformational macromolecules?8. Why is it important that weak forces, not strong forces, medi-ate biomolecular recognition?9. Why does the central role of weak forces in biomolecular inter-actions restrict living systems to a narrow range of environmentalconditions?10. Describe what is meant by the phrase “cells are steady-state systems.”32 Chapter 1 ● Chemistry Is the Logic of Biological PhenomenaFURTHER READINGAlberts, B., Bray, D., Lewis, J., et al., 1989. Molecular Biology of the Cell,2nd ed. New York: Garland Press.Goodsell, D. S., 1991. Inside a living cell. Trends in Biochemical Sciences16:203–206.Koonin, E. V., et al., 1996. Sequencing and analysis of bacterialgenomes.Current Biology 6:404–416.Lloyd, C., ed., 1986. Cell organization. Trends in Biochemical Sciences11:437–485.Loewy, A. G., Siekevitz, P., Menninger, J. R., Gallant, J. A. N., 1991.Cell Structure and Function. Philadelphia: Saunders College Publishing.Pace, N. R., 1996. New perspective on the natural microbial world:Molecular microbial ecology. ASM News 62:463–470.Service, R. F., 1997. Microbiologists explore life’s rich, hidden king-doms. Science 275:1740–1742.Solomon, E. P., Berg, L. R., Martin, D. W., and Villee, C., 1999. Biology,5th ed. Philadelphia: Saunders College Publishing.Wald, G., 1964. The origins of life. Proceedings of the National Academyof Science, U.S.A. 52:595–611.Watson, J. D., Hopkins, N.H., Roberts, J. W., et al., 1987. MolecularBiology of the Gene, 4th ed. Menlo Park, CA: Benjamin/CummingsPublishing Co.Woese, C. R., 1996. Phylogenetic trees: Whither microbiology? CurrentBiology 6:1060–1063.Further Reading 33Chapter 2Water, pH, and IonicEquilibriaSome of the magic: Students and teacher view a coral crab in Graham’s Harbour, San SalvadorIsland, the Bahamas. (Lara Call)Water is a major chemical component of the earth’s surface. It is indispens-able to life. Indeed, it is the only liquid that most organisms ever encounter.We alternately take it for granted because of its ubiquity and bland nature ormarvel at its many unusual and fascinating properties. At the center of this fas-cination is the role of water as the medium of life. Life originated, evolved,and thrives in the seas. Organisms invaded and occupied terrestrial and aerialniches, but none gained true independence from water. Typically, organismsare constituted of 70 to 90% water. Indeed, normal metabolic activity can occuronly when cells are at least 65% H2O. This dependency of life on water is nota simple matter, but it can be grasped through a consideration of the unusualchemical and physical properties of H2O. Subsequent chapters establish thatwater and its ionization products, hydrogen ions and hydroxide ions, are crit-OUTLINE2.1 ● Properties of Water2.2 ● pH2.3 ● Buffers2.4 ● Water’s Unique Role in the Fitness ofthe Environment“If there is magic on this planet, it is con-tained in water.”LOREN EISLEYInscribed on the wall of the NationalAquarium in Baltimore, MD34ical determinants of the structure and function of proteins, nucleic acids, andmembranes. In yet another essential role, water is an indirect participant—adifference in the concentration of hydrogen ions on opposite sides of a mem-brane represents an energized condition essential to biological mechanisms ofenergy transformation. First, let’s review the remarkable properties of water.2.1 ● Properties of WaterUnusual PropertiesIn comparison with chemical compounds of similar atomic organization andmolecular size, water displays unexpected properties. For example, comparewater, the hydride of oxygen, with hydrides of oxygen’s nearest neighbors inthe periodic table, namely, ammonia (NH3) and hydrogen fluoride (HF), orwith the hydride of its nearest congener, sulfur (H2S). Water has a substantiallyhigher boiling point, melting point, heat of vaporization, and surface tension.Indeed, all of these physical properties are anomalously high for a substanceof this molecular weight that is neither metallic nor ionic. These propertiessuggest that intermolecular forces of attraction between H2O molecules arehigh. Thus, the internal cohesion of this substance is high. Furthermore, waterhas an unusually high dielectric constant, its maximum density is found in theliquid (not the solid) state, and it has a negative volume of melting (that is,the solid form, ice, occupies more space than does the liquid form, water). Itis truly remarkable that so many eccentric properties should occur together ina single substance. As chemists, we expect to find an explanation for theseapparent anomalies in the structure of water. The key to its intermolecularattractions must lie in its atomic constitution. Indeed, the unrivaled ability to formhydrogen bonds is the crucial fact to understanding its properties.Structure of WaterThe two hydrogen atoms of water are linked covalently to oxygen, each shar-ing an electron pair, to give a nonlinear arrangement (Figure 2.1). This “bent”structure of the H2O molecule is of enormous significance to its properties. IfH2O were linear, it would be a nonpolar substance. In the bent configuration,however, the electronegative O atom and the two H atoms form a dipole thatrenders the molecule distinctly polar. Furthermore, this structure is ideallysuited to H-bond formation. Water can serve as both an H donor and an Hacceptor in H-bond formation. The potential to form four H bonds per watermolecule is the source of the strong intermolecular attractions that endow thissubstance with its anomalously high boiling point, melting point, heat of vapor-ization, and surface tension. In ordinary ice, the common crystalline form ofwater, each H2O molecule has four nearest neighbors to which it is hydrogenbonded: each H atom donates an H bond to the O of a neighbor, while the Oatom serves as an H-bond acceptor from H atoms bound to two different watermolecules (Figure 2.2). A local tetrahedral symmetry results.Hydrogen bonding in water is cooperative. That is, an H-bonded watermolecule serving as an acceptor is a better H-bond donor than an unbondedmolecule (and an H2O molecule serving as an H-bond donor becomes a bet-ter H-bond acceptor). Thus, participation in H bonding by H2O molecules isa phenomenon of mutual reinforcement. The H bonds between neighboringmolecules are weak (23 kJ/mol each) relative to the HOO covalent bonds (420kJ/mol). As a consequence, the hydrogen atoms are situated asymmetrically2.1 ● Properties of Water 35FIGURE 2.1 ● The structure of water. Twolobes of negative charge formed by the lone-pair electrons of the oxygen atom lie aboveand below the plane of the diagram. This elec-tron density contributes substantially to thelarge dipole moment and polarizability of thewater molecule. The dipole moment of watercorresponds to the OOH bonds having 33%ionic character. Note that the HOOOH angleis 104.3°, not 109°, the angular value found inmolecules with tetrahedral symmetry, such asCH4. Many of the important properties ofwater derive from this angular value, such asthe decreased density of its crystalline state, ice.(The dipole moment in this figure points inthe direction from negative to positive, the con-vention used by physicists and physicalchemists; organic chemists draw it pointing inthe opposite direction.)between the two oxygen atoms along the OOO axis. There is never any ambi-guity about which O atom the H atom is chemically bound to, nor to which Oit is H-bonded.Structure of IceIn ice, the hydrogen bonds form a space-filling, three-dimensional network.These bonds are directional and straight; that is, the H atom lies on a directline between the two O atoms. This linearity and directionality mean that theresultant H bonds are strong. In addition, the directional preference of the Hbonds leads to an open lattice structure. For example, if the water moleculesare approximated as rigid spheres centered at the positions of the O atoms inthe lattice, then the observed density of ice is actually only 57% of that expectedfor a tightly packed arrangement of such spheres. The H bonds in ice hold thewater molecules apart. Melting involves breaking some of the H bonds thatmaintain the crystal structure of ice so that the molecules of water (now liq-uid) can actually pack closer together. Thus, the density of ice is slightly lessthan the density of water. Ice floats, a property of great importance to aquaticorganisms in coldclimates.In liquid water, the rigidity of ice is replaced by fluidity, and the crystallineperiodicity of ice gives way to spatial homogeneity. The H2O molecules in liq-uid water form a random, H-bonded network with each molecule having anaverage of 4.4 close neighbors situated within a center-to-center distance of0.284 nm (2.84 Å). At least half of the hydrogen bonds have nonideal orien-tations (that is, they are not perfectly straight); consequently, liquid H2O lacksthe regular latticelike structure of ice. The space about an O atom is not definedby the presence of four hydrogens, but can be occupied by other water mole-36 Chapter 2 ● Water, pH, and Ionic EquilibriaFIGURE 2.2 ● The structure of normal ice. The hydrogen bonds in ice form a three-dimensional network. The smallest number of H2O molecules in any closed circuit of H-bonded molecules is six, so that this structure bears the name hexagonal ice. Covalentbonds are represented as solid lines, whereas hydrogen bonds are shown as dashed lines.The directional preference of H bonds leads to a rather open lattice structure for crys-talline water and, consequently, a low density for the solid state. The distance betweenneighboring oxygen atoms linked by a hydrogen bond is 0.274 nm. Because the covalentHOO bond is 0.995 nm, the HOO hydrogen bond length in ice is 0.18 nm.cules randomly oriented so that the local environment, over time, is essentiallyuniform. Nevertheless, the heat of melting for ice is but a small fraction (13%)of the heat of sublimation for ice (the energy needed to go from the solid tothe vapor state). This fact indicates that the majority of H bonds between H2Omolecules survive the transition from solid to liquid. At 10°C, 2.3 H bonds perH2O molecule remain, and the tetrahedral bond order persists even thoughsubstantial disorder is now present.Molecular Interactions in Liquid WaterThe present interpretation of water structure is that water molecules are con-nected by uninterrupted H bond paths running in every direction, spanningthe whole sample. The participation of each water molecule in an average stateof H bonding to its neighbors means that each molecule is connected to everyother in a fluid network of H bonds. The average lifetime of an H-bonded con-nection between two H2O molecules in water is 9.5 psec (picoseconds, where1 psec � 10�12 sec). Thus, about every 10 psec, the average H2O moleculemoves, reorients, and interacts with new neighbors, as illustrated in Figure 2.3.In summary, pure liquid water consists of H2O molecules held in a ran-dom, three-dimensional network that has a local preference for tetrahedralgeometry but contains a large number of strained or broken hydrogen bonds.The presence of strain creates a kinetic situation in which H2O molecules canswitch H-bond allegiances; fluidity ensues.Solvent PropertiesBecause of its highly polar nature, water is an excellent solvent for ionic sub-stances such as salts; nonionic but polar substances such as sugars, simple alco-hols, and amines; and carbonyl-containing molecules such as aldehydes andketones. Although the electrostatic attractions between the positive and nega-tive ions in the crystal lattice of a salt are very strong, water readily dissolvessalts. For example, sodium chloride is dissolved because dipolar water mole-cules participate in strong electrostatic interactions with the Na� and Cl� ions,leading to the formation of hydration shells surrounding these ions (Figure2.4). Although hydration shells are stable structures, they are also dynamic.Each water molecule in the inner hydration shell around a Na� ion is replacedon average every 2 to 4 nsec (nanoseconds, where 1 nsec � 10�9 sec) by anotherH2O. Consequently, a water molecule is trapped only several hundred timeslonger by the electrostatic force field of an ion than it is by the H-bonded net-work of water. (Recall that the average lifetime of H bonds between water mole-cules is about 10 psec.)Water Has a High Dielectric ConstantThe attractions between the water molecules interacting with, or hydrating,ions are much greater than the tendency of oppositely charged ions to attractone another. The ability of water to surround ions in dipole interactions and37FIGURE 2.3 ● The fluid network of H bonds linking water molecules in the liquid state.It is revealing to note that, in 10 psec, a photon of light (which travels at 3 � 108 m/sec)would move a distance of only 0.003 m.diminish their attraction for one another is a measure of its dielectric constant,D. Indeed, ionization in solution depends on the dielectric constant of the sol-vent; otherwise the strongly attracted positive and negative ions would unite toform neutral molecules. The strength of the dielectric constant is related tothe force, F, experienced between two ions of opposite charge separated by adistance, r, as given in the relationshipF � e1e2/Dr2where e1 and e2 are the charges on the two ions. Table 2.1 lists the dielectricconstants of some common liquids. Note that the dielectric constant for wateris more than twice that of methanol and more than 40 times that of hexane.Water Forms H Bonds with Polar SolutesIn the case of nonionic but polar compounds such as sugars, the excellent sol-vent properties of water stem from its ability to readily form hydrogen bondswith the polar functional groups on these compounds, such as hydroxyls,amines, and carbonyls. These polar interactions between solvent and solute arestronger than the intermolecular attractions between solute molecules causedby van der Waals forces and weaker hydrogen bonding. Thus, the solute mole-cules readily dissolve in water.Hydrophobic InteractionsThe behavior of water toward nonpolar solutes is different from the interac-tions just discussed. Nonpolar solutes (or nonpolar functional groups on bio-logical macromolecules) do not readily H bond to H2O, and, as a result, suchcompounds tend to be only sparingly soluble in water. The process of dissolv-ing such substances is accompanied by significant reorganization of the watersurrounding the solute so that the response of the solvent water to such solutescan be equated to “structure making.” Because nonpolar solutes must occupyspace, the random H-bond network of water must reorganize to accommodatethem. At the same time, the water molecules participate in as many H-bonded38 Chapter 2 ● Water, pH, and Ionic EquilibriaTable 2.1Dielectric Constants* of SomeCommon Solvents at 25°CSolvent Dielectric Constant (D)Water 78.5Methyl alcohol 32.6Ethyl alcohol 24.3Acetone 20.7Acetic acid 6.2Chloroform 5.0Benzene 2.3Hexane 1.9*The dielectric constant is also referred to as rel-ative permittivity by physical chemists.FIGURE 2.4 ● Hydration shells surroundingions in solution. Water molecules orient so thatthe electrical charge on the ion is sequesteredby the water dipole. For positive ions (cations),the partially negative oxygen atom of H2O istoward the ion in solution. Negatively chargedions (anions) attract the partially positivehydrogen atoms of water in creating theirhydration shells.interactions with one another as the temperature permits. Consequently, theH-bonded water network rearranges toward formation of a local cagelike(clathrate) structure surrounding each solute molecule (Figure 2.5). This fixedorientation of water molecules around a hydrophobic “solute” molecule resultsin a hydration shell. A major consequence of this rearrangement is that themolecules of H2O participating in the cage layer have markedly reduced ori-entational options. Water molecules tend to straddle the nonpolar solute suchthat two or three tetrahedral directions (H-bonding vectors) are tangential tothe space occupied by the inert solute. This“straddling” means that no waterH-bonding capacity is lost because no H-bond donor or acceptor of the H2Ois directed toward the caged solute. The water molecules forming theseclathrates are involved in highly ordered structures. That is, clathrate forma-tion is accompanied by significant ordering of structure or negative entropy.Under these conditions, nonpolar solute molecules experience a net attrac-tion for one another that is called hydrophobic interaction. The basis of thisinteraction is that when two nonpolar molecules meet, their joint solvationcage involves less surface area and less overall ordering of the water moleculesthan in their separate cages. The “attraction” between nonpolar solutes is anentropy-driven process due to a net decrease in order among the H2O mole-cules. To be specific, hydrophobic interactions between nonpolar moleculesare maintained not so much by direct interactions between the inert solutesthemselves as by the increase in entropy when the water cages coalesce andreorganize. Because interactions between nonpolar solute molecules and thewater surrounding them are of uncertain stoichiometry and do not share theequality of atom-to-atom participation implicit in chemical bonding, the termhydrophobic interaction is more correct than the misleading expression hydro-phobic bond.Amphiphilic MoleculesCompounds containing both strongly polar and strongly nonpolar groups arecalled amphiphilic molecules (from the Greek amphi meaning “both,” and phi-los meaning “loving”), also referred to as amphipathic molecules (from theGreek pathos meaning “passion”). Salts of fatty acids are a typical example that2.1 ● Properties of Water 39FIGURE 2.5 ● Formation of a clathrate struc-ture by water molecules surrounding ahydrophobic solute.amphiphilic molecules, amphipathic molecules ● compounds containing bothstrongly polar and strongly nonpolar groupshas biological relevance. They have a long nonpolar hydrocarbon tail and astrongly polar carboxyl head group, as in the sodium salt of palmitic acid(Figure 2.6). Their behavior in aqueous solution reflects the combination ofthe contrasting polar and nonpolar nature of these substances. The ionic car-boxylate function hydrates readily, whereas the long hydrophobic tail is intrin-sically insoluble. Nevertheless, sodium palmitate and other amphiphilic mole-cules readily disperse in water because the hydrocarbon tails of these substancesare joined together in hydrophobic interactions as their polar carboxylate func-tions are hydrated in typical hydrophilic fashion. Such clusters of amphipathicmolecules are termed micelles; Figure 2.7 depicts their structure. Of enormousbiological significance is the contrasting solute behavior of the two ends ofamphipathic molecules upon introduction into aqueous solutions. The polarends express their hydrophilicity in ionic interactions with the solvent, whereastheir nonpolar counterparts are excluded from the water into a hydrophobicdomain constituted from the hydrocarbon tails of many like molecules. It isthis behavior that accounts for the formation of membranes, the structuresthat define the limits and compartments of cells (see Chapter 9).40 Chapter 2 ● Water, pH, and Ionic EquilibriaFIGURE 2.6 ● An amphiphilic molecule:sodium palmitate. Amphiphilic molecules arefrequently symbolized by a ball and zig-zag linestructure, , where the ball represents thehydrophilic polar head and the zig-zag repre-sents the nonpolar hydrophobic hydrocarbontail. FIGURE 2.7 ● Micelle formation byamphiphilic molecules in aqueous solution.Negatively charged carboxylate head groupsorient to the micelle surface and interact withthe polar H2O molecules via H bonding. Thenonpolar hydrocarbon tails cluster in the inte-rior of the spherical micelle, driven byhydrophobic exclusion from the solvent andthe formation of favorable van der Waals inter-actions. Because of their negatively chargedsurfaces, neighboring micelles repel oneanother and thereby maintain a relative stabil-ity in solution.Influence of Solutes on Water PropertiesThe presence of dissolved substances disturbs the structure of liquid water sothat its properties change. The dynamic hydrogen-bonding pattern of watermust now accommodate the intruding substance. The net effect is that solutes,regardless of whether they are polar or nonpolar, fix nearby water moleculesin a more ordered array. Ions, by the establishment of hydration shells throughinteractions with the water dipoles, create local order. Hydrophobic effects, fordifferent reasons, make structures within water. To put it another way, by lim-iting the orientations that neighboring water molecules can assume, solutesgive order to the solvent and diminish the dynamic interplay among H2O mole-cules that occurs in pure water.Colligative PropertiesThis influence of the solute on water is reflected in a set of characteristicchanges in behavior that are termed colligative properties, or properties relatedby a common principle. These alterations in solvent properties are related inthat they all depend only on the number of solute particles per unit volumeof solvent and not on the chemical nature of the solute. These effects includefreezing point depression, boiling point elevation, vapor pressure lowering, andosmotic pressure effects. For example, 1 mol of an ideal solute dissolved in1000 g of water (a 1 m, or molal, solution) at 1 atm pressure depresses thefreezing point by 1.86°C, raises the boiling point by 0.543°C, lowers the vaporpressure in a temperature-dependent manner, and yields a solution whoseosmotic pressure relative to pure water is 22.4 atm. In effect, by imposing localorder on the water molecules, solutes make it more difficult for water to assumeits crystalline lattice (freeze) or escape into the atmosphere (boil or vaporize).Furthermore, when a solution (such as the 1 m solution discussed here) is sep-arated from a volume of pure water by a semipermeable membrane, the solu-tion draws water molecules across this barrier. The water molecules are mov-ing from a region of higher effective concentration (pure H2O) to a region oflower effective concentration (the solution). This movement of water into thesolution dilutes the effects of the solute that is present. The osmotic forceexerted by each mole of solute is so strong that it requires the imposition of22.4 atm of pressure to be negated (Figure 2.8).Osmotic pressure from high concentrations of dissolved solutes is a seri-ous problem for cells. Bacterial and plant cells have strong, rigid cell walls tocontain these pressures. In contrast, animal cells are bathed in extracellularfluids of comparable osmolarity, so no net osmotic gradient exists. Also, to min-imize the osmotic pressure created by the contents of their cytosol, cells tend2.1 ● Properties of Water 41FIGURE 2.8 ● The osmotic pressure of a 1molal (m) solution is equal to 22.4 atmospheresof pressure. (a) If a nonpermeant solute is sep-arated from pure water by a semipermeablemembrane through which H2O passes freely,(b) water molecules enter the solution (osmo-sis) and the height of the solution column inthe tube rises. The pressure necessary to pushwater back through the membrane at a rateexactly equaled by the water influx is theosmotic pressure of the solution. (c) For a 1 msolution, this force is equal to 22.4 atm of pres-sure. Osmotic pressure is directly proportionalto the concentration of the nonpermeantsolute.to store substances such as amino acids and sugars in polymeric form. For exam-ple, a molecule of glycogen or starch containing 1000 glucose units exerts only1/1000 the osmotic pressure that 1000 free glucose molecules would.Ionization of WaterWater shows a small but finite tendencyto form ions. This tendency is demon-strated by the electrical conductivity of pure water, a property that clearly estab-lishes the presence of charged species (ions). Water ionizes because the larger,strongly electronegative oxygen atom strips the electron from one of its hydro-gen atoms, leaving the proton to dissociate (Figure 2.9):Two ions are thus formed: protons or hydrogen ions, H�, and hydroxyl ions,OH�. Free protons are immediately hydrated to form hydronium ions, H3O�:Indeed, because most hydrogen atoms in liquid water are hydrogen-bonded to a neighboring water molecule, this protonic hydration is an instantaneousprocess and the ion products of water are H3O� and OH�:The amount of H3O� or OH� in 1 L (liter) of pure water at 25°C is 1 � 10�7mol; the concentrations are equal because the dissociation is stoichiometric.Although it is important to keep in mind that the hydronium ion, orhydrated hydrogen ion, represents the true state in solution, the conventionis to speak of hydrogen ion concentrations in aqueous solution, even though“naked” protons are virtually nonexistent. Indeed, H3O� itself attracts a hydra-tion shell by H bonding to adjacent water molecules to form an H9O4� species(Figure 2.10) and even more highly hydrated forms. Similarly, the hydroxylion, like all other highly charged species, is also hydrated.Proton JumpingBecause of the high degree of hydrogen bonding in water, H� ions show anapparent rate of migration in an electrical field that is vastly greater than otherunivalent cations in aqueous solution, such as Na� and K�. In effect, the nettransfer of a proton from molecule to molecule throughout the H-bonded net-work accounts for this apparent rapidity of migration (Figure 2.11).42 Chapter 2 ● Water, pH, and Ionic EquilibriaFIGURE 2.11 ● Proton jumping via the hydrogen-bonded network of water molecules.FIGURE 2.9 ● The ionization of water.FIGURE 2.10 ● The hydration of H3O�.Solid lines denote covalent bonds; dashed linesrepresent the H bonds formed between thehydronium ion and its waters of hydration.That is, the H-bonded network provides a natural route for rapid H� trans-port. This phenomenon of proton jumping thus occurs with little actual move-ment of the water molecules themselves. Ice has an electrical conductivity closeto that of water because such proton jumps also readily occur even when thewater molecules are fixed in a crystal lattice. Such conduction of protons viaH-bonded networks has been offered as an explanation for a number of rapidproton transfers of biological significance.Kw , the Ion Product of WaterThe dissociation of water into hydrogen ions and hydroxyl ions occurs to theextent that 10�7 mol of H� and 10�7 mol of OH� are present at equilibrium in1 L of water at 25°C.H2O 88n H� � OH�The equilibrium constant for this process isKeq �where brackets denote concentrations in moles per liter. Because the concen-tration of H2O in 1 L of pure water is equal to the number of grams in a literdivided by the gram molecular weight of H2O, or 1000/18, the molar con-centration of H2O in pure water is 55.5 M (molar). The decrease in H2O con-centration as a result of ion formation ([H�], [OH�] � 10�7 M) is negligiblein comparison, and thus its influence on the overall concentration of H2O canbe ignored. Thus,Keq � � 1.8 � 10�16Because the concentration of H2O in pure water is essentially constant, a newconstant, Kw, the ion product of water, can be written asKw � 55.5 Keq � 10�14 � [H�][OH�]The equation has the virtue of revealing the reciprocal relationship betweenH� and OH� concentrations of aqueous solutions. If a solution is acidic, thatis, of significant [H�], then the ion product of water dictates that the OH�concentration is correspondingly less. For example, if [H�] is 10�2 M, [OH�]must be 10�12 M (Kw � 10�14 � [10�2][OH�]; [OH�] � 10�12 M). Similarly,in an alkaline, or basic, solution in which [OH�] is great, [H�] is low.2.2 ● pHTo avoid the cumbersome use of negative exponents to express concentrationsthat range over 14 orders of magnitude, Sørensen, a Danish biochemist, devisedthe pH scale by defining pH as the negative logarithm of the hydrogen ion concen-tration1:pH � �log10 [H�](10�7)(10�7)55.5[H�][OH�][H2O]2.2 ● pH 431To be precise in physical chemical terms, the activities of the various components, not their molarconcentrations, should be used in these equations. The activity (a) of a solute component is definedas the product of its molar concentration, c, and an activity coefficient, �: a � [c]�. Most biochem-ical work involves dilute solutions, and the use of activities instead of molar concentrations is usu-ally neglected. However, the concentration of certain solutes may be very high in living cells.Table 2.2 gives the pH scale. Note again the reciprocal relationship between[H�] and [OH�]. Also, because the pH scale is based on negative logarithms,low pH values represent the highest H� concentrations (and the lowest OH�concentrations, as Kw specifies). Note also thatpKw � pH � pOH � 14The pH scale is widely used in biological applications because hydrogen ionconcentrations in biological fluids are very low, about 10�7 M or 0.0000001 M,a value more easily represented as pH 7. The pH of blood plasma, for exam-ple, is 7.4 or 0.00000004 M H�. Certain disease conditions may lower the plasmapH level to 6.8 or less, a situation that may result in death. At pH 6.8, the H�concentration is 0.00000016 M, four times greater than at pH 7.4.At pH 7, [H�] � [OH�]; that is, there is no excess acidity or basicity. Thepoint of neutrality is at pH 7, and solutions having a pH of 7 are said to be atneutral pH. The pH values of various fluids of biological origin or relevanceare given in Table 2.3. Because the pH scale is a logarithmic scale, two solu-tions whose pH values differ by one pH unit have a 10-fold difference in [H�].For example, grapefruit juice at pH 3.2 contains more than 12 times as muchH� as orange juice at pH 4.3.Dissociation of Strong ElectrolytesSubstances that are almost completely dissociated to form ions in solution arecalled strong electrolytes. The term electrolyte describes substances capable ofgenerating ions in solution and thereby causing an increase in the electricalconductivity of the solution. Many salts (such as NaCl and K2SO4) fit this cat-egory, as do strong acids (such as HCl) and strong bases (such as NaOH).Recall from general chemistry that acids are proton donors and bases are pro-44 Chapter 2 ● Water, pH, and Ionic EquilibriaTable 2.2pH ScaleThe hydrogen ion and hydroxyl ion concentrations are given in moles per liter at 25°C.pH [H�] [OH�]0 (100) 1.0 0.00000000000001 (10�14)1 (10�1) 0.1 0.0000000000001 (10�13)2 (10�2) 0.01 0.000000000001 (10�12)3 (10�3) 0.001 0.00000000001 (10�11)4 (10�4) 0.0001 0.0000000001 (10�10)5 (10�5) 0.00001 0.000000001 (10�9)6 (10�6) 0.000001 0.00000001 (10�8)7 (10�7) 0.0000001 0.0000001 (10�7)8 (10�8) 0.00000001 0.000001 (10�6)9 (10�9) 0.000000001 0.00001 (10�5)10 (10�10) 0.0000000001 0.0001 (10�4)11 (10�11) 0.00000000001 0.001 (10�3)12 (10�12) 0.000000000001 0.01 (10�2)13 (10�13) 0.0000000000001 0.1 (10�1)14 (10�14) 0.00000000000001 1.0 (100)Table 2.3The pH of Various Common FluidsFluid pHHousehold lye 13.6Bleach 12.6Household ammonia 11.4Milk of magnesia 10.3Baking soda 8.4Seawater 8.0Pancreatic fluid 7.8–8.0Blood plasma 7.4Intracellular fluidsLiver 6.9Muscle 6.1Saliva 6.6Urine 5–8Boric acid 5.0Beer 4.5Orange juice 4.3Grapefruit juice 3.2Vinegar 2.9Soft drinks 2.8Lemon juice 2.3Gastric juice 1.2–3.0Battery acid 0.35ton acceptors. In effect, the dissociationof a strong acid such as HCl in watercan be treated as a proton transfer reaction between the acid HCl and the baseH2O to give the conjugate acid H3O� and the conjugate base Cl�:HCl � H2O 88n H3O� � Cl�The equilibrium constant for this reaction isK �Customarily, because the term [H2O] is essentially constant in dilute aqueoussolutions, it is incorporated into the equilibrium constant K to give a new term,Ka, the acid dissociation constant (where Ka � K[H2O]). Also, the term [H3O�] is often replaced by H�, such thatKa �For HCl, the value of Ka is exceedingly large because the concentration of HClin aqueous solution is vanishingly small. Because this is so, the pH of HCl solu-tions is readily calculated from the amount of HCl used to make the solution:[H�] in solution � [HCl] added to solutionThus, a 1 M solution of HCl has a pH of 0; a 1 mM HCl solution has a pH of3. Similarly, a 0.1 M NaOH solution has a pH of 13. (Because [OH�] � 0.1 M,[H�] must be 10�13 M.)Viewing the dissociation of strong electrolytes another way, we see that theions formed show little affinity for one another. For example, in HCl in water,Cl� has very little affinity for H�:HCl 88n H� � Cl�and in NaOH solutions, Na� has little affinity for OH�. The dissociation ofthese substances in water is effectively complete.Dissociation of Weak ElectrolytesSubstances with only a slight tendency to dissociate to form ions in solutionare called weak electrolytes. Acetic acid, CH3COOH, is a good example:CH3COOH � H2O 88zy88 CH3COO� � H3O�The acid dissociation constant Ka for acetic acid is 1.74 � 10�5:Ka � � 1.74 � 10�5The term Ka is also called an ionization constant because it states the extentto which a substance forms ions in water. The relatively low value of Ka foracetic acid reveals that the un-ionized form, CH3COOH, predominates overH� and CH3COO� in aqueous solutions of acetic acid. Viewed another way,CH3COO�, the acetate ion, has a high affinity for H�.EXAMPLEWhat is the pH of a 0.1 M solution of acetic acid? Or, to restate the question,what is the final pH when 0.1 mol of acetic acid (HAc) is added to water andthe volume of the solution is adjusted to equal 1 L?[H�][CH3COO�][CH3COOH][H�][Cl�][HCl][H3O�][Cl�][H2O][HCl]2.2 ● pH 45ANSWERThe dissociation of HAc in water can be written simply as HAc 88zy88 H� � Ac�where Ac� represents the acetate ion, CH3COO�. In solution, some amountx of HAc dissociates, generating x amount of Ac� and an equal amount x ofH�. Ionic equilibria characteristically are established very rapidly. At equilib-rium, the concentration of HAc � Ac� must equal 0.1 M. So, [HAc] can berepresented as (0.1 � x) M, and [Ac�] and [H�] then both equal x molar.From 1.74 � 10�5 � ([H�][Ac�])/[HAc], we get 1.74 � 10�5 � x2/[0.1 � x].The solution to quadratic equations of this form (ax2 � bx � c � 0) is x � (�b� However, the calculation of x can be simplified by notingthat, because Ka is quite small, x �� 0.1 M. Therefore, Ka is essentially equalto x2/0.1. This simplification yields x2 � 1.74 � 10�6, or x � 1.32 � 10�3 Mand pH � 2.88.Henderson–Hasselbalch EquationConsider the ionization of some weak acid, HA, occurring with an acid disso-ciation constant, Ka. Then,HA 88zy88 H� � A�andKa �Rearranging this expression in terms of the parameter of interest, [H�], wehave[H�] �Taking the logarithm of both sides giveslog [H�] � log Ka � log10If we change the signs and define pKa � �log Ka, we havepH � pKa � log10orpH � pK a � log10This relationship is known as the Henderson–Hasselbalch equation. Thus, thepH of a solution can be calculated, provided Ka and the concentrations of theweak acid HA and its conjugate base A� are known. Note particularly that when[HA] � [A�], pH � pKa. For example, if equal volumes of 0.1 M HAc and 0.1M sodium acetate are mixed, thenpH � pKa � 4.76pKa � �log Ka � �log10 (1.74 � 10�5) � 4.76(Sodium acetate, the sodium salt of acetic acid, is a strong electrolyte and dis-sociates completely in water to yield Na� and Ac�.)[A�][HA][HA][A�][HA][A�]K a[HA][A�][H�][A�][HA]�b2 � 4ac)�2a.46 Chapter 2 ● Water, pH, and Ionic EquilibriaThe Henderson–Hasselbalch equation provides a general solution to thequantitative treatment of acid–base equilibria in biological systems. Table 2.4gives the acid dissociation constants and pKa values for some weak electrolytesof biochemical interest.EXAMPLEWhat is the pH when 100 mL of 0.1 N NaOH is added to 150 mL of 0.2 MHAc if pKa for acetic acid � 4.76?ANSWER100 mL 0.1 N NaOH � 0.01 mol OH�, which neutralizes 0.01 mol of HAc, giv-ing an equivalent amount of Ac�:OH� � HAc 88n Ac� � H2O0.02 mol of the original 0.03 mol of HAc remains essentially undissociated. Thefinal volume is 250 mL.pH � pKa � log10 � 4.76 � log (0.01 mol)/(0.02 mol)pH � 4.76 � log10 2 � 4.46If 150 mL of 0.2 M HAc had merely been diluted with 100 mL of water, thiswould leave 250 mL of a 0.12 M HAc solution. The pH would be given by:[Ac�][HAc]2.2 ● pH 47Table 2.4Acid Dissociation Constants and pKa Values for Some Weak Electrolytes (at 25°C)Acid Ka (M) pKaHCOOH (formic acid) 1.78 � 10�4 3.75CH3COOH (acetic acid) 1.74 � 10�5 4.76CH3CH2COOH (propionic acid) 1.35 � 10�5 4.87CH3CHOHCOOH (lactic acid) 1.38 � 10�4 3.86HOOCCH2CH2COOH (succinic acid) pK1* 6.16 � 10�5 4.21HOOCCH2CH2COO� (succinic acid) pK2 2.34 � 10�6 5.63H3PO4 (phosphoric acid) pK1 7.08 � 10�3 2.15H2PO4� (phosphoric acid) pK2 6.31 � 10�8 7.20HPO42� (phosphoric acid) pK3 3.98 � 10�13 12.40C3N2H5� (imidazole) 1.02 � 10�7 6.99C6O2N3H11� (histidine–imidazole group) pKR† 9.12 � 10�7 6.04H2CO3 (carbonic acid) pK1 1.70 � 10�4 3.77HCO3� (bicarbonate) pK2 5.75 � 10�11 10.24(HOCH2)3CNH3� (tris-hydroxymethyl aminomethane) 8.32 � 10�9 8.07NH4� (ammonium) 5.62 � 10�10 9.25CH3NH3� (methylammonium) 2.46 � 10�11 10.62*These pK values listed as pK1, pK2, or pK3 are in actuality pKa values for the respective disso-ciations. This simplification in notation is used throughout this book.†pKR refers to the imidazole ionization of histidine.Data from CRC Handbook of Biochemistry, The Chemical Rubber Co., 1968.Ka � � � 1.74 � 10�5x � 1.44 � 10�3 � [H�]pH � 2.84Clearly, the presence of sodium hydroxide has mostly neutralized the acidityof the acetic acid through formation of acetate ion.Titration CurvesTitration is the analytical method used to determine the amount of acid in asolution. A measured volume of the acid solution is titrated by slowly addinga solution of base, typically NaOH, of known concentration. As incrementalamounts of NaOH are added, the pH of the solution is determined and a plotof the pH of the solution versus the amount of OH� added yields a titrationcurve. The titration curve for acetic acid is shown in Figure 2.12. In consider-ing the progress of this titration, keep in mind two important equilibria:1. HAc 88zy88 H� � Ac� Ka � 1.74 � 10�52. H� � OH� 88n H2O K � � 5.55 � 1015As the titration begins, mostly HAc is present, plus some H� and Ac� inamounts that can be calculated (see the Example on page 45). Addition of asolution of NaOH allows hydroxide ions to neutralize any H� present. Note thatreaction (2) as written is strongly favored; its apparent equilibrium constant isgreater than 1015! As H� is neutralized, more HAc dissociates to H� and Ac�.As further NaOH is added, the pH gradually increases as Ac� accumulates atthe expense of diminishing HAc and the neutralization of H�. At the pointwhere half of the HAc has been neutralized, that is, where 0.5equivalent ofOH� has been added, the concentrations of HAc and Ac� are equal and pH �pKa for HAc. Thus, we have an experimental method for determining the pKavalues of weak electrolytes. These pKa values lie at the midpoint of their respec-tive titration curves. After all of the acid has been neutralized (that is, whenone equivalent of base has been added), the pH rises exponentially.The shapes of the titration curves of weak electrolytes are identical, asFigure 2.13 reveals. Note, however, that the midpoints of the different curvesvary in a way that characterizes the particular electrolytes. The pKa for aceticacid is 4.76, the pKa for imidazole is 6.99, and that for ammonium is 9.25.These pKa values are directly related to the dissociation constants of these sub-stances, or, viewed the other way, to the relative affinities of the conjugate basesfor protons. NH3 has a high affinity for protons compared to Ac�; NH4� is apoor acid compared to HAc.Phosphoric Acid Has Three Dissociable H�Figure 2.14 shows the titration curve for phosphoric acid, H3PO4. This sub-stance is a polyprotic acid, meaning it has more than one dissociable proton.Indeed, it has three, and thus three equivalents of OH� are required to neu-tralize it, as Figure 2.14 shows. Note that the three dissociable H� are lost indiscrete steps, each dissociation showing a characteristic pKa. Note that pK1occurs at pH � 2.15, and the concentrations of the acid H3PO4 and the con-jugate base H2PO4� are equal. As the next dissociation is approached, H2PO4�[H2O]K wx20.12 M[H�][Ac�][HAc]48 Chapter 2 ● Water, pH, and Ionic EquilibriaFIGURE 2.12 ● The titration curve for aceticacid. Note that the titration curve is relativelyflat at pH values near the pKa; in other words,the pH changes relatively little as OH� isadded in this region of the titration curve.is treated as the acid and HPO42� is its conjugate base. Their concentrationsare equal at pH 7.20, so pK2 � 7.20. (Note that at this point, 1.5 equivalentsof OH� have been added.) As more OH� is added, the last dissociable hydro-gen is titrated, and pK3 occurs at pH � 12.4, where [HPO42�] � [PO43�].A biologically important point is revealed by the basic shape of the titra-tion curves of weak electrolytes: in the region of the pKa, pH remains relativelyunaffected as increments of OH� (or H�) are added. The weak acid and itsconjugate base are acting as a buffer.2.2 ● pH 49FIGURE 2.13 ● The titration curves of sev-eral weak electrolytes: acetic acid, imidazole,and ammonium. Note that the shape of thesedifferent curves is identical. Only their positionalong the pH scale is displaced, in accordancewith their respective affinities for H� ions, asreflected in their differing pKa values.FIGURE 2.14 ● The titration curve for phosphoric acid. The chemical formulas showthe prevailing ionic species present at various pH values. Phosphoric acid (H3PO4) hasthree titratable hydrogens and therefore three midpoints are seen: at pH 2.15 (pK1), pH7.20 (pK2), and pH 12.4 (pK3).2.3 ● BuffersBuffers are solutions that tend to resist changes in their pH as acid or base isadded. Typically, a buffer system is composed of a weak acid and its conjugatebase. A solution of a weak acid that has a pH nearly equal to its pKa by defini-tion contains an amount of the conjugate base nearly equivalent to the weakacid. Note that in this region, the titration curve is relatively flat (Figure 2.15).Addition of H� then has little effect because it is absorbed by the followingreaction:H� � A� 88n HASimilarly, added OH� is consumed by the processOH� � HA 88n A� � H2OThe pH then remains relatively constant. The components of a buffer systemare chosen such that the pKa of the weak acid is close to the pH of interest. Itis at the pKa that the buffer system shows its greatest buffering capacity. At pHvalues more than one pH unit from the pKa, buffer systems become ineffec-tive because the concentration of one of the components is too low to absorbthe influx of H� or OH�. The molarity of a buffer is defined as the sum of theconcentrations of the acid and conjugate base forms.Maintenance of pH is vital to all cells. Cellular processes such as metabo-lism are dependent on the activities of enzymes, and in turn, enzyme activityis markedly influenced by pH, as the graphs in Figure 2.16 show. Consequently,changes in pH would be very disruptive to metabolism for reasons that becomeapparent in later chapters. Organisms have a variety of mechanisms to keepthe pH of their intra- and extracellular fluids essentially constant, but the pri-mary protection against harmful pH changes is provided by buffer systems.The buffer systems selected reflect both the need for a pKa value near pH 7and the compatibility of the buffer components with the metabolic machineryof cells. Two buffer systems act to maintain intracellular pH essentially con-stant—the phosphate (HPO42�/H2PO4�) system and the histidine system. ThepH of the extracellular fluid that bathes the cells and tissues of animals is main-tained by the bicarbonate/carbonic acid (HCO3�/H2CO3) system.50 Chapter 2 ● Water, pH, and Ionic EquilibriaFIGURE 2.15 ● A buffer system consists of aweak acid, HA, and its conjugate base, A�. ThepH varies only slightly in the region of the titra-tion curve where [HA] � [A�]. The unshadedbox denotes this area of greatest bufferingcapacity. Buffer action: when HA and A� areboth available in sufficient concentration, the solution can absorb input of either H�or OH�, and pH is maintained essentially constant.FIGURE 2.16 ● pH versus enzymatic activity. The activity of enzymes is very sensitive topH. The pH optimum of an enzyme is one of its most important characteristics. Pepsin isa protein-digesting enzyme active in the gastric fluid. Trypsin is also a proteolytic enzyme,but it acts in the more alkaline milieu of the small intestine. Lysozyme digests the cellwalls of bacteria; it is found in tears.Phosphate SystemThe phosphate system serves to buffer the intracellular fluid of cells at physi-ological pH because pK2 lies near this pH value. The intracellular pH of mostcells is maintained in the range between 6.9 and 7.4. Phosphate is an abun-dant anion in cells, both in inorganic form and as an important functionalgroup on organic molecules that serve as metabolites or macromolecular pre-cursors. In both organic and inorganic forms, its characteristic pK2 means thatthe ionic species present at physiological pH are sufficient to donate or accepthydrogen ions to buffer any changes in pH, as the titration curve for H3PO4in Figure 2.14 reveals. For example, if the total cellular concentration of phos-phate is 20 mM (millimolar) and the pH is 7.4, the distribution of the majorphosphate species is given bypH � pK2 � log107.4 � 7.20 � log10� 1.58Thus, if [HPO42�] � [H2PO4�] � 20 mM, then[HPO42�] � 12.25 mM and [H2PO4�] � 7.75 mMHistidine SystemHistidine is one of the 20 naturally occurring amino acids commonly found inproteins (see Chapter 4). It possesses as part of its structure an imidazole group,a five-membered heterocyclic ring possessing two nitrogen atoms. The pKa fordissociation of the imidazole hydrogen of histidine is 6.04.In cells, histidine occurs as the free amino acid, as a constituent of proteins,and as part of dipeptides in combination with other amino acids. Because theconcentration of free histidine is low and its imidazole pKa is more than 1 pHunit removed from prevailing intracellular pH, its role in intracellular buffer-ing is minor. However, protein-bound and dipeptide histidine may be the domi-nant buffering system in some cells. In combination with other amino acids,as inproteins or dipeptides, the imidazole pKa may increase substantially. Forexample, the imidazole pKa is 7.04 in anserine, a dipeptide containing �-alanine and histidine (Figure 2.17). Thus, this pKa is near physiological pH,and some histidine peptides are well suited for buffering at physiological pH.[HPO42�][H2PO4�][HPO42�][H2PO4�][HPO42�][H2PO4�]2.3 ● Buffers 51FIGURE 2.17 ● Anserine (N-�-alanyl-3-methyl-L-histidine) is an important dipeptidebuffer in the maintenance of intracellular pHin some tissues. The structure shown is the pre-dominant ionic species at pH 7. pK1 (COOH)� 2.64; pK2 (imidazole–N�H) � 7.04; pK3(NH3�) � 9.49.The Bicarbonate Buffer System of Blood PlasmaThe important buffer system of blood plasma is the bicarbonate/carbonic acidcouple:H2CO3 34 H� � HCO3�The relevant pKa, pK1 for carbonic acid, has a value far removed from the nor-mal pH of blood plasma (pH 7.4). (The pK1 for H2CO3 at 25°C is 3.77 (Table2.4), but at 37°C, pK1 is 3.57.) At pH 7.4, the concentration of H2CO3 is aminuscule fraction of the HCO3� concentration, and thus the plasma appearsto be poorly protected against an influx of OH� ions.pH � 7.4 � 3.57 � log10� 6761For example, if [HCO3�] � 24 mM, then [H2CO3] is only 3.55 M (3.55� 10�6 M), and an equivalent amount of OH� (its usual concentration inplasma) would swamp the buffer system, causing a dangerous rise in the plasmapH. How, then, can this bicarbonate system function effectively? The bicar-bonate buffer system works well because the critical concentration of H2CO3is maintained relatively constant through equilibrium with dissolved CO2 pro-duced in the tissues and available as a gaseous CO2 reservoir in the lungs.2“Good” BuffersNot many common substances have pKa values in the range from 6 to 8.Consequently, biochemists conducting in vitro experiments were limited in theirchoice of buffers effective at or near physiological pH. In 1966, N.E. Gooddevised a set of synthetic buffers to remedy this problem, and over the yearsthe list has expanded so that a “good” selection is available (Figure 2.18).[HCO3�][H2CO3][HCO3�][H2CO3]52 Chapter 2 ● Water, pH, and Ionic Equilibria2Well-fed human adults exhale about 1 kg of CO2 daily. Imagine the excretory problem if CO2were not a volatile gas!FIGURE 2.18 ● The pKa values and pH range of some “good” buffers.2.4 ● Water’s Unique Role in the Fitness of the EnvironmentThe remarkable properties of water render it particularly suitable to its uniquerole in living processes and the environment, and its presence in abundancefavors the existence of life. Let’s examine water’s physical and chemical prop-erties to see the extent to which they provide conditions that are advantageousto organisms.As a solvent, water is powerful yet innocuous. No other chemically inert sol-vent compares with water for the substances it can dissolve. Also, it is very impor-2.4 ● Water’s Unique Role in the Fitness of the Environment 53A D E E P E R L O O KHow the Bicarbonate Buffer System WorksGaseous carbon dioxide from the lungs and tissues is dissolved inthe blood plasma, symbolized as CO2(d), and hydrated to formH2CO3:CO2(g) 88zy88 CO2(d)CO2(d) � H2O 88zy88 H2CO3H2CO3 88zy88 H� � HCO3�Thus, the concentration of H2CO3 is itself buffered by the avail-able pools of CO2. The hydration of CO2 is actually mediated byan enzyme, carbonic anhydrase, which facilitates the equilibrium byrapidly catalyzing the reactionH2O � CO2(d) 88zy88 H2CO3Under the conditions of temperature and ionic strength prevail-ing in mammalian body fluids, the equilibrium for this reactionlies far to the left, such that about 500 CO2 molecules are presentin solution for every molecule of H2CO3. Because dissolved CO2and H2CO3 are in equilibrium, the proper expression for H2CO3availability is [CO2(d)] � [H2CO3], the so-called total carbonicacid pool, consisting primarily of CO2(d). The overall equilibriumfor the bicarbonate buffer system then isK hCO2(d) � H2O 34 H2CO3K aH2CO3 34 H� � HCO3�An expression for the ionization of H2CO3 under such conditions(that is, in the presence of dissolved CO2) can be obtained fromKh, the equilibrium constant for the hydration of CO2, and fromKa, the first acid dissociation constant for H2CO3:K h �Thus,[H2CO3] � K h[CO2(d)]Putting this value for [H2CO3] into the expression for the firstdissociation of H2CO3 givesKaTherefore, the overall equilibrium constant for the ionization ofH2CO3 in equilibrium with CO2(d) is given byKaKh �and KaKh, the product of two constants, can be defined as a newequilibrium constant, Koverall. The value of Kh is 0.003 at 37°Cand Ka, the ionization constant for H2CO3, is 10�3.57 � 0.000269.Therefore,Koverall � (0.000269)(0.003)� 8.07 � 10�7pKoverall � 6.1which yields the following Henderson–Hasselbalch relationship:pH � pKoverall � log10Although the prevailing blood pH of 7.4 is more than 1 pH unitaway from pKoverall, the bicarbonate system is still an effectivebuffer. That is, at blood pH, the concentration of the acid com-ponent of the buffer is less than 10% of the conjugate base com-ponent. One might imagine that this buffer component could beoverwhelmed by relatively small amounts of alkali, with conse-quent disastrous rises in blood pH. However, the acid componentis the total carbonic acid pool, that is, [CO2(d)] � [H2CO3],which is stabilized by its equilibrium with CO2(g). The gaseousCO2 buffers any losses from the total carbonic acid pool by enter-ing solution as CO2(d), and blood pH is effectively maintained.Thus, the bicarbonate buffer system is an open system. The naturalpresence of CO2 gas at a partial pressure of 40 mm Hg in thealveoli of the lungs and the equilibriumCO2(g) 88zy88 CO2(d)keep the concentration of CO2(d) (the principal component ofthe total carbonic acid pool in blood plasma) in the neighbor-hood of 1.2 mM. Plasma [HCO3�] is about 24 mM under suchconditions.[HCO3�][CO2(d)][H�][HCO3�][CO2(d)] �[H�][HCO3�]K h[CO2(d)] �[H�][HCO3�][H2CO3][H2CO3][CO2(d)]tant to life that water is a “poor” solvent for nonpolar substances. Thus, throughhydrophobic interactions, lipids coalesce, membranes form, boundaries arecreated delimiting compartments, and the cellular nature of life is established.Because of its very high dielectric constant, water is a medium for ionization.Ions enrich the living environment in that they enhance the variety of chemi-cal species and introduce an important class of chemical reactions. They pro-vide electrical properties to solutions and therefore to organisms. Aqueous solu-tions are the prime source of ions.The thermal properties of water are especially relevant to its environmentalfitness. It has great power as a buffer resisting thermal (temperature) change.Its heat capacity, or specific heat (4.1840 J/g°C), is remarkably high; it is tentimes greater than iron, five times greater than quartz or salt, and twice as greatas hexane. Its heat of fusion is 335 J/g. Thus, at 0°C, it takes a loss of 335 J tochange the state of 1 g of H2O from liquid to solid. Its heat of vaporization,2.24 kJ/g, is exceptionally high. These thermal properties mean that it takessubstantial changes in heat content to alter the temperature and especially thestate of water. Water’s thermal properties allow it to buffer the climate throughsuch processes as condensation, evaporation, melting, and freezing. Fur-thermore, these properties allow effective temperature regulation in livingorganisms. For example, heat generated within an organism as a result ofmetabolism can be efficiently eliminated by evaporationor conduction. Thethermal conductivity of water is very high in comparison with other liquids.The anomalous expansion of water as it cools to temperatures near its freez-ing point is a unique attribute of great significance to its natural fitness. Aswater cools, H bonding increases because the thermal motions of the mole-cules are lessened. Hydrogen bonding tends to separate the water molecules(Figure 2.2), and thus the density of water decreases. These changes in den-sity mean that, at temperatures below 4°C, cool water rises and, most impor-tantly, ice freezes on the surface of bodies of water, forming an insulating layerprotecting the liquid water underneath.Water has the highest surface tension (75 dyne/cm) of all common liquids(except mercury). Together, surface tension and density determine how higha liquid rises in a capillary system. Capillary movement of water plays a promi-nent role in the life of plants. Lastly, consider osmosis, the bulk movement ofwater in the direction from a dilute aqueous solution to a more concentratedone across a semipermeable boundary. Such bulk movements determine theshape and form of living things.Water is truly a crucial determinant of the fitness of the environment. Ina very real sense, organisms are aqueous systems in a watery world.54H U M A N B I O C H E M I S T R YBlood pH and RespirationHyperventilation, defined as a breathing rate more rapid thannecessary for normal CO2 elimination from the body, can resultin an inappropriately low [CO2(g)] in the blood. Central nervoussystem disorders such as meningitis, encephalitis, or cerebralhemorrhage, as well as a number of drug- or hormone-inducedphysiological changes, can lead to hyperventilation. As [CO2(g)]drops due to excessive exhalation, [H2CO3] in the blood plasmafalls, followed by decline in [H�] and [HCO3�] in the bloodplasma. Blood pH rises within 20 sec of the onset of hyperventi-lation, becoming maximal within 15 min. [H�] can change fromits normal value of 40 nM (pH � 7.4) to 18 nM (pH � 7.74). Thisrise in plasma pH (increase in alkalinity) is termed respiratoryalkalosis.Hypoventilation is the opposite of hyperventilation and ischaracterized by an inability to excrete CO2 rapidly enough tomeet physiological needs. Hypoventilation can be caused by nar-cotics, sedatives, anesthetics, and depressant drugs; diseases of thelung also lead to hypoventilation. Hypoventilation results in res-piratory acidosis, as CO2(g) accumulates, giving rise to H2CO3,which dissociates to form H� and HCO3�.PROBLEMS1. Calculate the pH of the following.a. 5 � 10�4 M HClb. 7 � 10�5 M NaOHc. 2 M HCld. 3 � 10�2 M KOHe. 0.04 mM HClf. 6 � 10�9 M HCl2. Calculate the following from the pH values given in Table 2.3.a. [H�] in vinegarb. [H�] in salivac. [H�] in household ammoniad. [OH�] in milk of magnesiae. [OH�] in beerf. [H�] inside a liver cell3. The pH of a 0.02 M solution of an acid was measured at 4.6.a. What is the [H�] in this solution?b. Calculate the acid dissociation constant Ka and pKa for thisacid.4. The Ka for formic acid is 1.78 � 10�4 M.a. What is the pH of a 0.1 M solution of formic acid?b. 150 mL of 0.1 M NaOH is added to 200 mL of 0.1 M formicacid, and water is added to give a final volume of 1 L. What is thepH of the final solution?5. Given 0.1 M solutions of acetic acid and sodium acetate, describethe preparation of 1 L of 0.1 M acetate buffer at a pH of 5.4.6. If the internal pH of a muscle cell is 6.8, what is the[HPO42�]/[H2PO4�] ratio in this cell?7. Given 0.1 M solutions of Na3PO4 and H3PO4, describe thepreparation of 1 L of a phosphate buffer at a pH of 7.5. What arethe molar concentrations of the ions in the final buffer solution,including Na� and H�?8. BICINE is a compound containing a tertiary amino groupwhose relevant pKa is 8.3 (Figure 2.18). Given 1 L of 0.05 MBICINE with its tertiary amino group in the unprotonated form,how much 0.1 N HCl must be added to have a BICINE buffer solu-tion of pH 7.5? What is the molarity of BICINE in the final buffer?9. What are the approximate fractional concentrations of the fol-lowing phosphate species at pH values of 0, 2, 4, 6, 8, 10, and 12?a. H3PO4b. H2PO4�c. HPO42�d. PO43�10. Citric acid, a tricarboxylic acid important in intermediarymetabolism, can be symbolized as H3A. Its dissociation reactions areH3A 34 H� � H2A� pK1 � 3.13H2A� 34 H� � HA2� pK2 � 4.76HA2� 34 H� � A3� pK3 � 6.40If the total concentration of the acid and its anion forms is 0.02M, what are the individual concentrations of H3A, H2A�, HA2�,and A3� at pH 5.2?11. a. If 50 mL of 0.01 M HCl is added to 100 mL of 0.05 M phos-phate buffer at pH 7.2, what is the resultant pH? What are theconcentrations of H2PO4� and HPO42� in the final solution?b. If 50 mL of 0.01 M NaOH is added to 100 mL of 0.05 M phos-phate buffer at pH 7.2, what is the resultant pH? What are theconcentrations of H2PO4� and HPO42� in this final solution?12. If the plasma pH is 7.4 and the plasma concentration ofHCO3� is 15 mM, what is the plasma concentration of H2CO3?What is the plasma concentration of CO2(dissolved)? If metabolicactivity changes the concentration of CO2(dissolved) to 3 mM, and[HCO3�] remains at 15 mM, what is the pH of the plasma?Further Reading 55FURTHER READINGBeynon, R. J., and Easterby, J. S., 1996. Buffer Solutions: The Basics. NewYork: IRL Press: Oxford University Press.Cooper, T. G., 1977. The Tools of Biochemistry, Chap. 1. New York: John Wiley& Sons.Darvey, I. G., and Ralston, G. B., 1993. Titration curves—misshapen ormislabeled? Trends in Biochemical Sciences 18:69–71.Edsall, J. T., and Wyman, J., 1958. Carbon dioxide and carbonic acid, inBiophysical Chemistry, Vol. 1, Chap. 10. New York: Academic Press.Franks, F., ed., 1982. The Biophysics of Water. New York: John Wiley & Sons.Henderson, L. J., 1913. The Fitness of the Environment. New York: MacmillanCo. (Republished 1970. Gloucester, MA: P. Smith).Hille, B., 1992. Ionic Channels of Excitable Membranes, 2nd ed., Chap. 10.Sunderland, MA: Sinauer Associates.Masoro, E. J., and Siegel, P. D., 1971. Acid–Base Regulation: Its Physiologyand Pathophysiology. Philadelphia: W.B. Saunders Co.Perrin, D. D., 1982. Ionization Constants of Inorganic Acids and Bases inAqueous Solution. New York: Pergamon Press.Rose, B. D., 1994. Clinical Physiology of Acid–Base and Electrolyte Disorders, 4thed., New York: McGraw–Hill, Inc.Segel, I. H., 1976. Biochemical Calculations, 2nd ed., Chap. 1. New York: JohnWiley & Sons.Stillinger, F. H., 1980. Water revisited. Science 209:451–457.Chapter 3Thermodynamics ofBiological SystemsOUTLINE3.1 ● Basic Thermodynamic Concepts3.2 ● The Physical Significance ofThermodynamic Properties3.3 ● The Effects of pH on Standard-StateFree Energies3.4 ● The Important Effect of Concentrationon Net Free Energy Changes3.5 ● The Importance of Coupled Processesin Living Things3.6 ● The High-Energy Biomolecules3.7 ● Complex Equilibria Involved in ATPHydrolysis3.8 ● The Daily Human Requirement for ATP56A theory is the more impressive thegreater is the simplicity of its premises,the more different are the kinds of thingsit relates and the more extended is itsrange of applicability. Therefore, thedeep impression which classical thermo-dynamics made upon me. It is the onlyphysical theory of universal contentwhich I am convinced, that within theframework of applicability of its basic con-cepts, will never be overthrown.ALBERT EINSTEINSun emblem of Louis XIV on a gate at Versailles. The sun is the primesource of energy for life, and thermodynamics is the gateway to under-standingmetabolism. (Giraudon/Art Research, New York)The activities of living things require energy. Movement, growth, synthesis ofbiomolecules, and the transport of ions and molecules across membranes alldemand energy input. All organisms must acquire energy from their sur-roundings and must utilize that energy efficiently to carry out life processes.To study such bioenergetic phenomena requires familiarity with thermody-namics, a collection of laws and principles describing the flows and inter-changes of heat, energy, and matter in systems of interest. Thermodynamicsalso allows us to determine whether or not chemical processes and reactionsoccur spontaneously. The student should appreciate the power and practicalvalue of thermodynamic reasoning and realize that this is well worth the effortneeded to understand it.Even the most complicated aspects of thermodynamics are based ultimatelyon three rather simple and straightforward laws. These laws and their exten-sions sometimes run counter to our intuition. However, once truly understood,the basic principles of thermodynamics become powerful devices for sortingout complicated chemical and biochemical problems. At this milestone in ourscientific development, thermodynamic thinking becomes an enjoyable andsatisfying activity.Several basic thermodynamic principles are presented in this chapter,including the analysis of heat flow, entropy production, and free energy func-tions and the relationship between entropy and information. In addition, someancillary concepts are considered, including the concept of standard states, theeffect of pH on standard-state free energies, the effect of concentration on thenet free energy change of a reaction, and the importance of coupled processesin living things. The chapter concludes with a discussion of ATP and otherenergy-rich compounds.3.1 ● Basic Thermodynamic ConceptsIn any consideration of thermodynamics, a distinction must be made betweenthe system and the surroundings. The system is that portion of the universewith which we are concerned, whereas the surroundings include everythingelse in the universe (Figure 3.1). The nature of the system must also be speci-fied. There are three basic systems: isolated, closed, and open. An isolated sys-tem cannot exchange matter or energy with its surroundings. A closed systemmay exchange energy, but not matter, with the surroundings. An open systemmay exchange matter, energy, or both with the surroundings. Living things aretypically open systems that exchange matter (nutrients and waste products)and heat (from metabolism, for example) with their surroundings.The First Law: Heat, Work, and Other Forms of EnergyIt was realized early in the development of thermodynamics that heat could beconverted into other forms of energy, and moreover that all forms of energycould ultimately be converted to some other form. The first law of thermo-dynamics states that the total energy of an isolated system is conserved. Thermo-dynamicists have formulated a mathematical function for keeping track of heattransfers and work expenditures in thermodynamic systems. This function iscalled the internal energy, commonly designated E or U. The internal energydepends only on the present state of a system and hence is referred to as astate function. The internal energy does not depend on how the system gotthere and is thus independent of path. An extension of this thinking is that wecan manipulate the system through any possible pathway of changes, and aslong as the system returns to the original state, the internal energy, E, will nothave been changed by these manipulations.The internal energy, E, of any system can change only if energy flows in orout of the system in the form of heat or work. For any process that convertsone state (state 1) into another (state 2), the change in internal energy, �E, isgiven as�E � E2 � E1 � q � w (3.1)where the quantity q is the heat absorbed by the system from the surroundings, andw is the work done on the system by the surroundings. Mechanical work is defined3.1 ● Basic Thermodynamic Concepts 57FIGURE 3.1 ● The characteristics of isolated,closed, and open systems. Isolated systemsexchange neither matter nor energy with theirsurroundings. Closed systems may exchangeenergy, but not matter, with their surroundings.Open systems may exchange either matter orenergy with the surroundings.Isolated systemNo exchange of matter or heatIsolated systemSurroundingsClosed systemHeat exchange may occurClosed systemSurroundingsHeat Open systemHeat exchange and/or matter exchange may occurOpen systemSurroundingsHeat Matter as movement through some distance caused by the application of a force. Both of thesemust occur for work to have occurred. For example, if a person strains to lifta heavy weight but fails to move the weight at all, then, in the thermodynamicsense, no work has been done. (The energy expended in the muscles of thewould-be weight lifter is given off in the form of heat.) In chemical and bio-chemical systems, work is often concerned with the pressure and volume of thesystem under study. The mechanical work done on the system is defined as w � �P�V, where P is the pressure and �V is the volume change and is equalto V2 � V1. When work is defined in this way, the sign on the right side ofEquation (3.1) is positive. (Sometimes w is defined as work done by the system;in this case, the equation is �E � q � w.) Work may occur in many forms, suchas mechanical, electrical, magnetic, and chemical. �E, q, and w must all havethe same units. The calorie, abbreviated cal, and kilocalorie (kcal), have beentraditional choices of chemists and biochemists, but the SI unit, the joule, isnow recommended.Enthalpy: A More Useful Function for Biological SystemsIf the definition of work is limited to mechanical work, an interesting simpli-fication is possible. In this case, �E is merely the heat exchanged at constant vol-ume. This is so because if the volume is constant, no mechanical work can bedone on or by the system. Then �E � q. Thus �E is a very useful quantity inconstant volume processes. However, chemical and especially biochemicalprocesses and reactions are much more likely to be carried out at constantpressure. In constant pressure processes, �E is not necessarily equal to the heattransferred. For this reason, chemists and biochemists have defined a functionthat is especially suitable for constant pressure processes. It is called theenthalpy, H, and it is defined asH � E � PV (3.2)The clever nature of this definition is not immediately apparent. However, ifthe pressure is constant, then we have�H � �E � P�V � q � w � P�V � q � P�V � P�V � q (3.3)Clearly, �H is equal to the heat transferred in a constant pressure process.Often, because biochemical reactions normally occur in liquids or solids ratherthan in gases, volume changes are small and enthalpy and internal energy are oftenessentially equal.In order to compare the thermodynamic parameters of different reactions,it is convenient to define a standard state. For solutes in a solution, the stan-dard state is normally unit activity (often simplified to 1 M concentration).Enthalpy, internal energy, and other thermodynamic quantities are often givenor determined for standard-state conditions and are then denoted by a super-script degree sign (“°”), as in �H°, �E°, and so on.Enthalpy changes for biochemical processes can be determined experi-mentally by measuring the heat absorbed (or given off) by the process in acalorimeter (Figure 3.2). Alternatively, for any process A zy B at equilibrium, thestandard-state enthalpy change for the process can be determined from the tem-perature dependence of the equilibrium constant:58 Chapter 3 ● Thermodynamics of Biological SystemsFIGURE 3.2 ● Diagram of a calorimeter. The reaction vessel is completely submerged ina water bath. The heat evolved by a reaction is determined by measuring the rise in tem-perature of the water bath.ReactionvesselSamplecupWater bathin calorimeterchamberChamberJacketChamberthermometerIgnitionelectrodes Jacketthermometer�H° � �R (3.4)Here R is the gas constant, defined as R � 8.314 J/mol �K. A plot of R(ln Keq)versus 1/T is called a van’t Hoff plot.EXAMPLEIn a study1 of the temperature-induced reversible denaturation of the proteinchymotrypsinogen,Native state (N) 34 denatured state (D)Keq � [D]/[N]John F. Brandts measured the equilibrium constants for the denaturation overa range of pH and temperatures. The data for pH 3:T(K): 324.4 326.1 327.5 329.0 330.7 332.0 333.8Keq: 0.041 0.12 0.27 0.68 1.9 5.0 21A plot of R(ln Keq) versus 1/T (a van’t Hoff plot) is shown in Figure 3.3. �H°for the denaturation process at any temperature is the negative of the slope ofthe plot at that temperature. As shown, �H° at 54.5°C (327.5 K) is�H° � �[�3.2 � (�17.6)]/[(3.04 � 3.067) � 10�3] � �533 kJ/molWhat does this value of �H° mean for the unfolding of the protein? Positivevalues of �H° would be expected for the breaking of hydrogen bonds as wellas for the exposure of hydrophobic groups from the interior of the native,folded protein during the unfolding process. Such events would raise theenergy of the protein–water solution. The magnitude of this enthalpy change(533 kJ/mol) at 54.5°C is large, compared to similar values of �H° for otherproteins and for this same protein at 25°C (Table 3.1). If we consider only thispositive enthalpy change for the unfolding process, the native, folded state isstrongly favored. As we shall see, however, other parameters must be taken intoaccount.d(ln Keq)d(1�T)3.1 ● Basic Thermodynamic Concepts 591Brandts, J. F., 1964. The thermodynamics of protein denaturation. I. The denaturation of chy-motrypsinogen. Journal of the American Chemical Society 86:4291–4301.Table 3.1Thermodynamic Parameters for Protein DenaturationProtein �H° �S ° �G ° �Cp(and conditions) kJ/mol kJ/mol � K kJ/mol kJ/mol � KChymotrypsinogen 164 �0.440 31.0 10.9(pH 3, 25°C)�-Lactoglobulin �88 �0.300 2.5 9.0(5 M urea, pH 3, 25°C)Myoglobin 180 �0.400 57.0 5.9(pH 9, 25°C)Ribonuclease 240 �0.780 3.8 8.4(pH 2.5, 30°C)Adapted from Cantor, C., and Schimmel, P., 1980. Biophysical Chemistry. San Francisco: W.H.Freeman, and Tanford, C., 1968. Protein denaturation. Advances in Protein Chemistry 23:121–282.FIGURE 3.3 ● The enthalpy change, �H°, for a reaction can be determined from the slope of a plot of R ln K eq versus 1/T. To illus-trate the method, the values of the data pointson either side of the 327.5 K (54.5°C) datapoint have been used to calculate �H° at54.5°C. Regression analysis would normally bepreferable. (Adapted from Brandts, J. F., 1964. The thermo-dynamics of protein denaturation. I. The denaturation of chy-motrypsinogen. Journal of the American Chemical Society86:4291–4301.)30R ln Keq2.98 3.04100020100–10–20–303.00 3.02 3.06 3.08 3.1054.5°C–3.21–(–17.63)= 14.423.04–3.067= –0.027(K–1)TThe Second Law and Entropy: An Orderly Way of Thinking About DisorderThe second law of thermodynamics has been described and expressed in manydifferent ways, including the following.1. Systems tend to proceed from ordered (low entropy or low probability) statesto disordered (high entropy or high probability) states.2. The entropy of the system plus surroundings is unchanged by reversibleprocesses; the entropy of the system plus surroundings increases for irreversibleprocesses.3. All naturally occurring processes proceed toward equilibrium, that is, to astate of minimum potential energy.Several of these statements of the second law invoke the concept of entropy,which is a measure of disorder and randomness in the system (or the sur-roundings). An organized or ordered state is a low-entropy state, whereas a dis-ordered state is a high-entropy state. All else being equal, reactions involvinglarge, positive entropy changes, �S, are more likely to occur than reactions forwhich �S is not large and positive.Entropy can be defined in several quantitative ways. If W is the number ofways to arrange the components of a system without changing the internal60 Chapter 3 ● Thermodynamics of Biological SystemsA D E E P E R L O O KEntropy, Information, and the Importance of “Negentropy”When a thermodynamic system undergoes an increase in entropy,it becomes more disordered. On the other hand, a decrease inentropy reflects an increase in order. A more ordered system ismore highly organized and possesses a greater information con-tent. To appreciate the implications of decreasing the entropy ofa system, consider the random collection of letters in the figure.This disorganized array of letters possesses no inherent informa-tion content, and nothing can be learned by its perusal. On theother hand, this particular array of letters can be systematicallyarranged to construct the first sentence of the Einstein quotationthat opened this chapter: “A theory is the more impressive thegreater is the simplicity of its premises, the more different are thekinds of things it relates and the more extended is its range ofapplicability.”Arranged in this way, this same collection of 151 letters pos-sesses enormous information content—the profound words of agreat scientist. Just as it would have required significant effort torearrange these 151 letters in this way, so large amounts of energyare required to construct and maintain living organisms. Energyinput is required to produce information-rich, organized struc-tures such as proteins and nucleic acids. Information content canbe thought of as negative entropy. In 1945 Erwin Schrödinger tooktime out from his studies of quantum mechanics to publish adelightful book entitled What is Life? In it, Schrödinger coinedthe term negentropy to describe the negative entropy changes thatconfer organization and information content to living organisms.Schrödinger pointed out that organisms must “acquire negen-tropy” to sustain life.ahetoryisthemoreimpressivetehgre ateristehsimplicityofitspremisesthemoerdifferentar ethhethingskdofinsitrelatesa ndthem oreexte ndedisitsrangeofapplicabilityAenergy or enthalpy (that is, the number of microscopic states at a given tem-perature, pressure, and amount of material), then the entropy is given byS � k ln W (3.5)where k is Boltzmann’s constant (k � 1.38 � 10�23 J/K). This definition is use-ful for statistical calculations (it is in fact a foundation of statistical thermody-namics), but a more common form relates entropy to the heat transferred ina process:dSreversible � (3.6)where dSreversible is the entropy change of the system in a reversible2 process,q is the heat transferred, and T is the temperature at which the heat transferoccurs.The Third Law: Why Is “Absolute Zero” So Important?The third law of thermodynamics states that the entropyof any crystalline, per-fectly ordered substance must approach zero as the temperature approaches0 K, and at T � 0 K entropy is exactly zero. Based on this, it is possible to estab-lish a quantitative, absolute entropy scale for any substance asS � CPd ln T (3.7)where CP is the heat capacity at constant pressure. The heat capacity of any sub-stance is the amount of heat one mole of it can store as the temperature ofthat substance is raised by one degree. For a constant pressure process, this isdescribed mathematically asCP � (3.8)If the heat capacity can be evaluated at all temperatures between 0 K and thetemperature of interest, an absolute entropy can be calculated. For biologicalprocesses, entropy changes are more useful than absolute entropies. The entropychange for a process can be calculated if the enthalpy change and free energychange are known.Free Energy: A Hypothetical but Useful DeviceAn important question for chemists, and particularly for biochemists, is, “Willthe reaction proceed in the direction written?” J. Willard Gibbs, one of thefounders of thermodynamics, realized that the answer to this question lay in acomparison of the enthalpy change and the entropy change for a reaction ata given temperature. The Gibbs free energy, G, is defined asG � H � TS (3.9)For any process A 34 B at constant pressure and temperature, the free energychange is given by�G � �H � T�S (3.10)dHdT�T0dqT3.1 ● Basic Thermodynamic Concepts 612A reversible process is one that can be reversed by an infinitesimal modification of a variable.If �G is equal to 0, the process is at equilibrium, and there is no net flow eitherin the forward or reverse direction. When �G � 0, �S � �H/T, and theenthalpic and entropic changes are exactly balanced. Any process with anonzero �G proceeds spontaneously to a final state of lower free energy. If �Gis negative, the process proceeds spontaneously in the direction written. If �Gis positive, the reaction or process proceeds spontaneously in the reverse direc-tion. (The sign and value of �G do not allow us to determine how fast theprocess will go.) If the process has a negative �G, it is said to be exergonic,whereas processes with positive �G values are endergonic.The Standard-State Free Energy ChangeThe free energy change, �G, for any reaction depends upon the nature of thereactants and products, but it is also affected by the conditions of the reaction,including temperature, pressure, pH, and the concentrations of the reactantsand products. As explained earlier, it is useful to define a standard state forsuch processes. If the free energy change for a reaction is sensitive to solutionconditions, what is the particular significance of the standard-state free energychange? To answer this question, consider a reaction between two reactants Aand B to produce the products C and D.A � B 34 C � D (3.11)The free energy change for non–standard-state concentrations is given by�G � �G° � RT ln (3.12)At equilibrium, �G � 0 and [C][D]/[A][B] � Keq. We then have�G ° � �RT ln Keq (3.13)or, in base 10 logarithms,�G ° � �2.3RT log10 Keq (3.14)This can be rearranged toKeq � 10��G°/2.3RT (3.15)In any of these forms, this relationship allows the standard-state free energychange for any process to be determined if the equilibrium constant is known.More importantly, it states that the equilibrium established for a reaction in solutionis a function of the standard-state free energy change for the process. That is, �G° isanother way of writing an equilibrium constant.EXAMPLEThe equilibrium constants determined by Brandts at several temperatures forthe denaturation of chymotrypsinogen (see previous Example) can be used tocalculate the free energy changes for the denaturation process. For example,the equilibrium constant at 54.5°C is 0.27, so�G° � �(8.314 J/mol �K)(327.5 K) ln (0.27)�G° � �(2.72 kJ/mol) ln (0.27)�G ° � 3.56 kJ/molThe positive sign of �G ° means that the unfolding process is unfavorable; thatis, the stable form of the protein at 54.5°C is the folded form. On the otherhand, the relatively small magnitude of �G ° means that the folded form is onlyslightly favored. Figure 3.4 shows the dependence of �G° on temperature for[C][D][A][B]62 Chapter 3 ● Thermodynamics of Biological Systemsthe denaturation data at pH 3 (from the data given in the Example on page59).Having calculated both �H° and �G ° for the denaturation of chy-motrypsinogen, we can also calculate �S°, using Equation (3.10):�S° � � (3.16)At 54.5°C (327.5 K),�S° � �(3560 � 533,000 J/mol)/327.5 K�S° � 1,620 J/mol �KFigure 3.5 presents the dependence of �S° on temperature for chymotryp-sinogen denaturation at pH 3. A positive �S° indicates that the protein solu-tion has become more disordered as the protein unfolds. Comparison of thevalue of 1.62 kJ/mol �K with the values of �S° in Table 3.1 shows that the pre-sent value (for chymotrypsinogen at 54.5°C) is quite large. The physical sig-nificance of the thermodynamic parameters for the unfolding of chymotryp-sinogen becomes clear in the next section.3.2 ● The Physical Significance of Thermodynamic PropertiesWhat can thermodynamic parameters tell us about biochemical events? Thebest answer to this question is that a single parameter (�H or �S, for exam-ple) is not very meaningful. A positive �H° for the unfolding of a protein mightreflect either the breaking of hydrogen bonds within the protein or the expo-sure of hydrophobic groups to water (Figure 3.6). However, comparison of sev-eral thermodynamic parameters can provide meaningful insights about a process. Forexample, the transfer of Na� and Cl� ions from the gas phase to aqueous solu-tion involves a very large negative �H° (thus a very favorable stabilization ofthe ions) and a comparatively small �S° (Table 3.2). The negative entropy termreflects the ordering of water molecules in the hydration shells of the Na� andCl� ions. This unfavorable effect is more than offset by the large heat of hydra-tion, which makes the hydration of ions a very favorable process overall. Thenegative entropy change for the dissociation of acetic acid in water also reflectsthe ordering of water molecules in the ion hydration shells. In this case, how-ever, the enthalpy change is much smaller in magnitude. As a result, �G° for(�G° � �H°)T3.2 ● The Physical Significance of Thermodynamic Properties 63FIGURE 3.4 ● The dependence of �G° ontemperature for the denaturation of chy-motrypsinogen. (Adapted from Brandts, J. F., 1964. Thethermodynamics of protein denaturation. I. The denaturation ofchymotrypsinogen. Journal of the American ChemicalSociety 86:4291–4301.)FIGURE 3.5 ● The dependence of �S° ontemperature for the denaturation of chy-motrypsinogen. (Adapted from Brandts, J. F., 1964. Thethermodynamics of protein denaturation. I. The denatura-tion of chymotrypsinogen. Journal of the American ChemicalSociety 86:4291– 4301.)FIGURE 3.6 ● Unfolding of a soluble proteinexposes significant numbers of nonpolargroups to water, forcing order on the solventand resulting in a negative �S° for the unfold-ing process. Yellow spheres represent nonpolargroups; blue spheres are polar and/or chargedgroups.10∆G, kJ/mol50 52Temperature, °C86420–2–4–6–8–1054 56 58 60 622.452Temperature, °C2.32.22.12.01.91.81.71.61.51.4∆S, kJ/mol•K54 56 58 60Folded Unfolded dissociation of acetic acid in water is positive, and acetic acid is thus a weak(largely undissociated) acid.The transfer of a nonpolar hydrocarbon molecule from its pure liquid towater is anappropriate model for the exposure of protein hydrophobic groupsto solvent when a protein unfolds. The transfer of toluene from liquid tolueneto water involves a negative �S°, a positive �G°, and a �H° that is small com-pared to �G° (a pattern similar to that observed for the dissociation of aceticacid). What distinguishes these two very different processes is the change in heat capac-ity (Table 3.2). A positive heat capacity change for a process indicates that themolecules have acquired new ways to move (and thus to store heat energy). Anegative �CP means that the process has resulted in less freedom of motionfor the molecules involved. �CP is negative for the dissociation of acetic acidand positive for the transfer of toluene to water. The explanation is that polarand nonpolar molecules both induce organization of nearby water molecules,but in different ways. The water molecules near a nonpolar solute are organizedbut labile. Hydrogen bonds formed by water molecules near nonpolar solutesrearrange more rapidly than the hydrogen bonds of pure water. On the otherhand, the hydrogen bonds formed between water molecules near an ion areless labile (rearrange more slowly) than they would be in pure water. Thismeans that �CP should be negative for the dissociation of ions in solution, asobserved for acetic acid (Table 3.2).3.3 ● The Effect of pH on Standard-State Free EnergiesFor biochemical reactions in which hydrogen ions (H�) are consumed or pro-duced, the usual definition of the standard state is awkward. Standard state forthe H� ion is 1 M, which corresponds to pH 0. At this pH, nearly all enzymeswould be denatured, and biological reactions could not occur. It makes moresense to use free energies and equilibrium constants determined at pH 7.Biochemists have thus adopted a modified standard state, designated withprime (�) symbols, as in �G°�, K�eq, �H°�, and so on. For values determinedin this way, a standard state of 10�7 M H� and unit activity (1 M for solutions,1 atm for gases and pure solids defined as unit activity) for all other compo-nents (in the ionic forms that exist at pH 7) is assumed. The two standardstates can be related easily. For a reaction in which H� is produced,A 88n B� � H� (3.17)64 Chapter 3 ● Thermodynamics of Biological SystemsTable 3.2Thermodynamic Parameters for Several Simple Processes*�H° �S ° �G ° �CPProcess kJ/mol kJ/mol � K kJ/mol kJ/mol � KHydration of ions†Na�(g) � Cl�(g) 88n Na�(aq) � Cl�(aq) �760.0 �0.185 �705.0Dissociation of ions in solution‡H2O � CH3COOH 88n H3O� � CH3COO� 0�10.3 �0.126 000027.26 �0.143Transfer of hydrocarbon from pure liquid to water‡Toluene (in pure toluene) 88n toluene (aqueous) 1.72 �0.071 000022.7 000.265*All data collected for 25°C.†Berry, R. S., Rice, S. A., and Ross, J., 1980. Physical Chemistry. New York: John Wiley.‡Tanford, C., 1980. The Hydrophobic Effect. New York: John Wiley.the relation of the equilibrium constants for the two standard states isK eq� � Keq[H�] (3.18)and �G °� is given by�G°� � �G° � RT ln [H�] (3.19)For a reaction in which H� is consumed,A� � H� 88n B (3.20)the equilibrium constants are related byKeq� � (3.21)and �G°� is given by�G°� � �G° � RT ln � �G° � RT ln [H�] (3.22)3.4 ● The Important Effect of Concentration on Net Free Energy ChangesEquation (3.12) shows that the free energy change for a reaction can be verydifferent from the standard-state value if the concentrations of reactants andproducts differ significantly from unit activity (1 M for solutions). The effectscan often be dramatic. Consider the hydrolysis of phosphocreatine:Phosphocreatine � H2O 88n creatine � Pi (3.23)This reaction is strongly exergonic and �G° at 37°C is �42.8 kJ/mol. Physiologi-cal concentrations of phosphocreatine, creatine, and inorganic phosphate arenormally between 1 mM and 10 mM. Assuming 1 mM concentrations and usingEquation (3.12), the �G for the hydrolysis of phosphocreatine is�G � �42.8 kJ/mol � (8.314 J/mol � K)(310 K) ln (3.24)�G � �60.5 kJ/mol (3.25)At 37°C, the difference between standard-state and 1 mM concentrations forsuch a reaction is thus approximately �17.7 kJ/mol.3.5 ● The Importance of Coupled Processes in Living ThingsMany of the reactions necessary to keep cells and organisms alive must runagainst their thermodynamic potential, that is, in the direction of positive �G.Among these are the synthesis of adenosine triphosphate and other high-energymolecules and the creation of ion gradients in all mammalian cells. Theseprocesses are driven in the thermodynamically unfavorable direction via cou-pling with highly favorable processes. Many such coupled processes are discussedlater in this text. They are crucially important in intermediary metabolism,oxidative phosphorylation, and membrane transport, as we shall see.We can predict whether pairs of coupled reactions will proceed sponta-neously by simply summing the free energy changes for each reaction. Forexample, consider the reaction from glycolysis (discussed in Chapter 19) �[0.001][0.001][0.001] �� 1[H�]�Keq[H�]3.5 ● The Importance of Coupled Processes in Living Things 65involving the conversion of phospho(enol)pyruvate (PEP) to pyruvate (Figure3.7). The hydrolysis of PEP is energetically very favorable, and it is used to drivephosphorylation of ADP to form ATP, a process that is energetically unfavor-able. Using values of �G that would be typical for a human erythrocyte:PEP � H2O 88n pyruvate � Pi �G � �78 kJ/mol (3.26)ADP � Pi 88n ATP � H2O �G � �55 kJ/mol (3.27)PEP � ADP 88n pyruvate � ATP Total �G � �23 kJ/mol (3.28)The net reaction catalyzed by this enzyme depends upon coupling between thetwo reactions shown in Equations (3.26) and (3.27) to produce the net reac-tion shown in Equation (3.28) with a net negative �G°�. Many other examplesof coupled reactions are considered in our discussions of intermediary metab-olism (Part III). In addition, many of the complex biochemical systems dis-cussed in the later chapters of this text involve reactions and processes withpositive �G°� values that are driven forward by coupling to reactions with anegative �G°�.3.6 ● The High-Energy BiomoleculesVirtually all life on earth depends on energy from the sun. Among life forms,there is a hierarchy of energetics: certain organisms capture solar energydirectly, whereas others derive their energy from this group in subsequentprocesses. Organisms that absorb light energy directly are called phototrophicorganisms. These organisms store solar energy in the form of various organicmolecules. Organisms that feed on these latter molecules, releasing the storedenergy in a series of oxidative reactions, are called chemotrophic organisms.Despite these differences, both types of organisms share common mechanismsfor generating a useful form of chemical energy. Once captured in chemicalform, energy can be released in controlled exergonic reactions to drive a vari-ety of life processes (which require energy). A small family of universal bio-molecules mediates the flow of energy from exergonic reactions to the energy-requiring processes of life. These molecules are the reduced coenzymes and thehigh-energy phosphate compounds. Phosphate compounds are considered highenergy if they exhibit large negative free energies of hydrolysis (that is, if �G°�is more negative than �25 kJ/mol).Table 3.3 lists the most important members of the high-energy phosphatecompounds. Such molecules include phosphoric anhydrides (ATP, ADP), an enolphosphate (PEP), an acyl phosphate (acetyl phosphate), and a guanidino phosphate(creatine phosphate). Also included are thioesters, such as acetyl-CoA, whichdo not contain phosphorus, but which have a high free energy of hydrolysis.As noted earlier in this chapter, the exact amount of chemical free energy avail-able from the hydrolysis of such compounds depends on concentration, pH,temperature, and so on, but the �G°� values for hydrolysis of these substancesare substantially more negative than for most other metabolic species. Twoimportant points: first, high-energy phosphate compounds are not long-termenergy storage substances. They are transient forms of stored energy, meant tocarry energy from point to point, from one enzyme system to another, in theminute-to-minute existence of the cell. (As we shall see in subsequent chap-ters, other molecules bear the responsibility for long-term storage of energy66 Chapter 3 ● Thermodynamics of Biological SystemsFIGURE 3.7 ● The pyruvate kinase reaction.COO-OPO32-CCH2PEPCOO-ADP + Pi ATPC OCH3Pyruvate3.6 ● The High-Energy Biomolecules 67Table 3.3Free Energies of Hydrolysis of Some High-Energy Compounds*�G °�Compound (and Hydrolysis Product) (kJ/mol) StructurePhosphoenolpyruvate (pyruvate � Pi) �62.23�,5�-Cyclic adenosine monophosphate (5�-AMP) �50.41,3-Bisphosphoglycerate (3-phosphoglycerate � Pi) �49.6Creatine phosphate (creatine � Pi) �43.3Acetyl phosphate (acetate � Pi) �43.3Adenosine-5�-triphosphate (ADP � Pi) �35.7†Adenosine-5�-triphosphate (ADP � Pi), excess Mg2� �30.5Adenosine-5�-diphosphate (AMP � Pi) �35.7–2O3PC COCH2OO–O–NNNH2CH2 OH HHHO OHPOO 3'5'NNC COO PO32–OHHCH2O–2O3P–2O3P NHCNCH2COO–CH3+NH2COOPO32–CH3–OO– O– O– NNNH2CH2 OH HHHOHOHOPOOPOOPONN–OO– O–NNNH2CH2 OH HHHOHOHOPOOPONN(continued)supplies.) Second, the term high-energy compound should not be construed toimply that these molecules are unstable and hydrolyze or decompose unpre-dictably. ATP, for example, is quite a stable molecule. A substantial activationenergy must be delivered to ATP to hydrolyze the terminal, or , phosphategroup. In fact, as shown in Figure 3.8, the activation energy that must be absorbedby the molecule to break the OOP bond is normally 200 to 400 kJ/mol, whichis substantially larger than the net 30.5 kJ/mol released in the hydrolysis reac-tion. Biochemists are much more concerned with the net release of 30.5 kJ/mol68 Chapter 3 ● Thermodynamics of Biological SystemsTable 3.3 Continued�G°�Compound (and Hydrolysis Product) (kJ/mol) StructurePyrophosphate (Pi � Pi) in 5 mM Mg2� �33.6Adenosine-5�-triphosphate (AMP � PPi), excess Mg2� �32.3 (See ATP structure on previous page)Uridine diphosphoglucose (UDP � glucose) �31.9Acetyl-coenzyme A (acetate � CoA) �31.5NHNCH2 OH HHHOHOHOPOO–OPOO–OOOHHOH HH OHHHOCH2OHOAdenineCH2OH HHHO OHOPOO–O OPOO–CH2 CH3CH3CCHOHC NHOCH2 CH2 C NHOCH2 CH2 S CH3CONNNH2CH2 OH HHHOHOHSCH3+–OOCCHCH2CH2NH3+NNO–POO–OPOO––OS-adenosylmethionine (methionine � adenosine) �25.6‡than with the activation energy for the reaction (because suitable enzymes copewith the latter). The net release of large quantities of free energy distinguishesthe high-energy phosphoric anhydrides from their “low-energy” ester cousins,such as glycerol-3-phosphate (Table 3.3). The next section provides a quanti-tative framework for understanding these comparisons.3.6 ● The High-Energy Biomolecules 69HOCH2 OH HOHHOHOHCH2 O PO32–HOCH2 OH HOHHOHOHCH2 O PO32–�G °�Compound (and Hydrolysis Product) (kJ/mol) StructureLower-Energy Phosphate CompoundsGlucose-1-P (glucose � Pi) �21.0Fructose-1-P (fructose � Pi) �16.0Glucose-6-P (glucose � Pi) �13.9sn-Glycerol-3-P (glycerol � Pi) �9.2Adenosine-5�-monophosphate (adenosine � Pi) �9.2OHHHOH HH OHHHOCH2OO–2O3PCOHHCH2O–2O3P CH2OHCH2 OH HHHOHOHOPOO––ONNH2NNN*Adapted primarily from Handbook of Biochemistry and Molecular Biology, 1976, 3rd ed. In Physical and Chemical Data,G. Fasman, ed., Vol. 1, pp. 296–304. Boca Raton, FL: CRC Press.†From Gwynn, R. W., and Veech, R. L., 1973. The equilibrium constants of the adenosine triphosphate hydrolysis andthe adenosine triphosphate-citrate lyase reactions. Journal of Biological Chemistry 248:6966–6972.‡From Mudd, H., and Mann, J., 1963. Activation of methionine for transmethylation. Journal of Biological Chemistry238:2164–2170.ATP Is an Intermediate Energy-Shuttle MoleculeOne last point about Table 3.3 deserves mention. Given the central importanceof ATP as a high-energy phosphate in biology, students are sometimes surprisedto find that ATP holds an intermediate place in the rank of high-energy phos-phates. PEP, cyclic AMP, 1,3-BPG, phosphocreatine, acetyl phosphate, andpyrophosphate all exhibit higher values of �G°�. This is not a biological anom-aly. ATP is uniquely situated between the very high energy phosphates synthe-sized in the breakdown of fuel molecules and the numerous lower-energy accep-tor molecules that are phosphorylated in the course of further metabolicreactions. ADP can accept both phosphates and energy from the higher-energyphosphates, and the ATP thus formed can donate both phosphates and energyto the lower-energy molecules of metabolism. The ATP/ADP pair is an inter-mediately placed acceptor/donor system among high-energy phosphates. In this context, ATP functions as a very versatile but intermediate energy-shuttle device that interacts with many different energy-coupling enzymes ofmetabolism.Group Transfer PotentialMany reactions in biochemistry involve the transfer of a functional group froma donor molecule to a specific receptor molecule or to water. The concept ofgroup transfer potential explains the tendency for such reactions to occur.Biochemists define the group transfer potential as the free energy change thatoccurs upon hydrolysis, that is, upon transfer of the particular group to water.This concept and its terminology are preferable to the more qualitative notionof high-energy bonds.The concept of group transfer potential is not particularly novel. Otherkinds of transfer (of hydrogen ions and electrons, for example) are commonly70 Chapter 3 ● Thermodynamics of Biological SystemsFIGURE 3.8 ● The activation energies for phosphoryl group-transfer reactions (200 to400 kJ/mol) are substantially larger than the free energy of hydrolysis of ATP (�30.5kJ/mol).Activation energy≅ 200–400kJmolReactantsProductsTransition statePhosphoryl grouptransfer potential≅ –30.5 kJ/molP+ADPATPcharacterized in terms of appropriate measures of transfer potential (pKa andreduction potential, �o, respectively). As shown in Table 3.4, the notion ofgroup transfer is fully analogous to those of ionization potential and reductionpotential. The similarity is anything but coincidental, because all of these arereally specific instances of free energy changes. If we writeAH 88n A� � H� (3.29a)we really don’t mean that a proton has literally been removed from the acidAH. In the gas phase at least, this would require the input of approximately1200 kJ/mol! What we really mean is that the proton has been transferred to asuitable acceptor molecule, usually water:AH � H2O 88n A� � H3O� (3.29b)The appropriate free energy relationship is of coursepKa � (3.30)Similarly, in the caseof an oxidation-reduction reactionA 88n A� � e� (3.31a)we don’t really mean that A oxidizes independently. What we really mean (andwhat is much more likely in biochemical systems) is that the electron is trans-ferred to a suitable acceptor:A � H� 88n A� � H2 (3.31b)and the relevant free energy relationship is��o � (3.32)where n is the number of equivalents of electrons transferred, and � is Faraday’sconstant.Similarly, the release of free energy that occurs upon the hydrolysis of ATPand other “high-energy phosphates” can be treated quantitatively in terms ofgroup transfer. It is common to write for the hydrolysis of ATPATP � H2O 88n ADP � Pi (3.33)��G °n�12�G °2.303 RT3.6 ● The High-Energy Biomolecules 71Table 3.4Types of Transfer PotentialProton Transfer Standard Reduction Potential Group TransferPotential (Electron Transfer Potential(Acidity) Potential) (High-Energy Bond)Simple equation AH 34 A� � H� A 34 A� � e� A � P 34 A � PiEquation including acceptor AH � H2O 34 A � H� 34 A � PO42� � H2O 34A� � H3O� A� � H2 AOOH � HPO42�Measure of transfer potential pK a � ��o � ln K eq �Free energy change �G ° per mole of �G ° per mole of �G° per mole ofof transfer is given by: H� transferred e� transferred phosphate transferredAdapted from: Klotz, I. M., 1986. Introduction to Biomolecular Energetics. New York: Academic Press.��G°RT��G°n��G°2.303 RT12The free energy change, which we henceforth call the group transfer potential,is given by�G° � �RT ln Keq (3.34)where Keq is the equilibrium constant for the group transfer, which is normallywritten asKeq � (3.35)Even this set of equations represents an approximation, because ATP, ADP, andPi all exist in solutions as a mixture of ionic species. This problem is discussedin a later section. For now, it is enough to note that the free energy changeslisted in Table 3.3 are the group transfer potentials observed for transfers towater.Phosphoric Acid AnhydridesATP contains two pyrophosphoryl or phosphoric acid anhydride linkages, as shownin Figure 3.9. Other common biomolecules possessing phosphoric acid anhy-dride linkages include ADP, GTP, GDP and the other nucleoside triphosphates,sugar nucleotides such as UDP-glucose, and inorganic pyrophosphate itself. Allexhibit large negative free energies of hydrolysis, as shown in Table 3.3. Thechemical reasons for the large negative �G°� values for the hydrolysis reactionsinclude destabilization of the reactant due to bond strain caused by electro-static repulsion, stabilization of the products by ionization and resonance, andentropy factors due to hydrolysis and subsequent ionization.Destabilization Due to Electrostatic RepulsionElectrostatic repulsion in the reactants is best understood by comparing thesephosphoric anhydrides with other reactive anhydrides, such as acetic anhy-dride. As shown in Figure 3.10a, the electronegative carbonyl oxygen atomswithdraw electrons from the CPO bonds, producing partial negative chargeson the oxygens and partial positive charges on the carbonyl carbons. Each of[ADP][Pi][ATP][H2O]72 Chapter 3 ● Thermodynamics of Biological SystemsPOHNH2OO–POO–O POO–O CH2OHON NNNPhosphoric anhydridelinkagesO–OATP(adenosine-5'-triphosphate)FIGURE 3.9 ● The triphosphate chain of ATP contains two pyrophosphate linkages,both of which release large amounts of energy upon hydrolysis.these electrophilic carbonyl carbons is further destabilized by the other acetylgroup, which is also electron-withdrawing in nature. As a result, acetic anhy-dride is unstable with respect to the products of hydrolysis.The situation with phosphoric anhydrides is similar. The phosphorus atomsof the pyrophosphate anion are electron-withdrawing and destabilize PPi withrespect to its hydrolysis products. Furthermore, the reverse reaction, reforma-tion of the anhydride bond from the two anionic products, requires that theelectrostatic repulsion between these anions be overcome (see following).Stabilization of Hydrolysis Products by Ionization and ResonanceThe pyrophosphate moiety possesses three negative charges at pH values above7.5 or so (note the pKa values, Figure 3.10a). The hydrolysis products, two mole-3.6 ● The High-Energy Biomolecules 732 CH3CCCH3O–OCOO OH3C2Acetic anhydride: POROO–POOR' O–Phosphoric anhydrides:PO–O– P–OO–OR'ROPOO–POO– OH O–pK1 = 0.8pK2 = 2.0pK3 = 6.7pK4 = 9.4+H2OH+H2O+Most likely formbetween pH 6.7and 9.4(a)δ+ δ+δ– δ–OO OO(b)Competing resonance in acetic anhydrideCO–H3C O+COCH3COH3C OCOCH3COH3C O+CO–CH3These can only occur alternatelySimultaneous resonance in the hydrolysis productsCOH3C O– –OCOCH3CO–H3C OC–OCH3OThese resonances can occur simultaneouslyPyrophosphate:FIGURE 3.10 ● (a) Electrostatic repulsion between adjacent partialpositive charges (on carbon and phosphorus, respectively) is relievedupon hydrolysis of the anhydride bonds of acetic anhydride and phos-phoric anhydrides. The predominant form of pyrophosphate at pHvalues between 6.7 and 9.4 is shown. (b) The competing resonancesof acetic anhydride and the simultaneous resonance forms of thehydrolysis product, acetate.cules of inorganic phosphate, both carry about two negative charges, at pHvalues above 7.2. The increased ionization of the hydrolysis products helps tostabilize the electrophilic phosphorus nuclei.Resonance stabilization in the products is best illustrated by the reactantanhydrides (Figure 3.10b). The unpaired electrons of the bridging oxygenatoms in acetic anhydride (and phosphoric anhydride) cannot participate inresonance structures with both electrophilic centers at once. This competingresonance situation is relieved in the product acetate or phosphate molecules.Entropy Factors Arising from Hydrolysis and IonizationFor the phosphoric anhydrides, and for most of the high-energy compoundsdiscussed here, there is an additional “entropic” contribution to the free energyof hydrolysis. Most of the hydrolysis reactions of Table 3.3 result in an increasein the number of molecules in solution. As shown in Figure 3.11, the hydroly-sis of ATP (as pH values above 7) creates three species—ADP, inorganic phos-phate (Pi), and a hydrogen ion—from only two reactants (ATP and H2O). Theentropy of the solution increases because the more particles, the more disor-dered the system.3 (This effect is ionization-dependent because, at low pH, the74 Chapter 3 ● Thermodynamics of Biological Systems3Imagine the “disorder” created by hitting a crystal with a hammer and breaking it into many smallpieces.FIGURE 3.11 ● Hydrolysis of ATP to ADP(and/or of ADP to AMP) leads to relief of elec-trostatic repulsion. Pδ+ O–CH2OOδ––O OOHPδ+ Pδ+O– O–Oδ– Oδ–OOHNH2NNN NOCH2OHOOHNH2NNN NCH2OHOOHNH2NNN NP OHO–O–O +P OHO–O–O +Pδ+ O–Oδ–OO–OOδ––O Pδ+Pδ+ O–Oδ–OOH2OH2O+H++H+ATPADPAMPhydrogen ion created in many of these reactions simply protonates one of thephosphate oxygens, and one fewer “particle” results from the hydrolysis.)A Comparison of the Free Energy of Hydrolysis of ATP, ADP, and AMPThe concepts of destabilization of reactants and stabilization of productsdescribedfor pyrophosphate also apply for ATP and other phosphoric anhy-drides (Figure 3.11). ATP and ADP are destabilized relative to the hydrolysisproducts by electrostatic repulsion, competing resonance, and entropy. AMP,on the other hand, is a phosphate ester (not an anhydride) possessing only asingle phosphoryl group and is not markedly different from the product inor-ganic phosphate in terms of electrostatic repulsion and resonance stabilization.Thus, the �G°� for hydrolysis of AMP is much smaller than the correspondingvalues for ATP and ADP.Phosphoric–Carboxylic AnhydridesThe mixed anhydrides of phosphoric and carboxylic acids, frequently calledacyl phosphates, are also energy-rich. Two biologically important acyl phos-phates are acetyl phosphate and 1,3-bisphosphoglycerate. Hydrolysis of thesespecies yields acetate and 3-phosphoglycerate, respectively, in addition to inor-ganic phosphate (Figure 3.12). Once again, the large �G°� values indicate thatthe reactants are destabilized relative to products. This arises from bond strain,which can be traced to the partial positive charges on the carbonyl carbon andphosphorus atoms of these structures. The energy stored in the mixed anhy-dride bond (which is required to overcome the charge–charge repulsion) isreleased upon hydrolysis. Increased resonance possibilities in the products rel-ative to the reactants also contribute to the large negative �G°� values. The3.6 ● The High-Energy Biomolecules 75FIGURE 3.12 ● The hydrolysis reactions of acetyl phosphate and 1,3-bisphospho-glycerate.OO–OPO–O HCOH+CH2CO–PO–OOO–O HCOHCH2CO–PO–OO+ O–OPO–HO +1,3–Bisphosphoglycerate 3–Phosphoglycerate ∆G°' = –49.6 kJ/molO–OPO–O +CCH3 O–O CCH3 + O–OPO–HO +∆G°' = –43.3 kJ/molO H2O H+H2O H+Acetyl phosphatevalue of �G°� depends on the pKa values of the starting anhydride and theproduct phosphoric and carboxylic acids, and of course also on the pH of themedium.Enol PhosphatesThe largest value of �G°� in Table 3.3 belongs to phosphoenolpyruvate or PEP,an example of an enolic phosphate. This molecule is an important interme-diate in carbohydrate metabolism and, due to its large negative �G°�, it is apotent phosphorylating agent. PEP is formed via dehydration of 2-phospho-glycerate by enolase during fermentation and glycolysis. PEP is subsequentlytransformed into pyruvate upon transfer of its phosphate to ADP by pyruvatekinase (Figure 3.13). The very large negative value of �G°� for the latter reac-tion is to a large extent the result of a secondary reaction of the enol form ofpyruvate. Upon hydrolysis, the unstable enolic form of pyruvate immediatelyconverts to the keto form with a resulting large negative �G°� (Figure 3.14).Together, the hydrolysis and subsequent tautomerization result in an overall �G°�of �62.2 kJ/mol.76 Chapter 3 ● Thermodynamics of Biological SystemsFIGURE 3.13 ● Phosphoenolpyruvate (PEP) is produced by the enolase reaction (in gly-colysis; see Chapter 19) and in turn drives the phosphorylation of ADP to form ATP inthe pyruvate kinase reaction.FIGURE 3.14 ● Hydrolysis and the subsequent tautomerization account for the verylarge �G°� of PEP.ATPO–OPO–OCHOHCOO– C COO–C H2CH2CH2CCOO– CH3C COO–O2–Phosphoglycerate Phosphoenolpyruvate(PEP)PhosphoenolpyruvatePEPPyruvate Mg2+, K+ PyruvatekinaseEnolase Mg2+ H2OO–OPO–OO–OPO–OH+ADP++OHPO–O∆G = –28.6 kJ/mol O–∆G = –33.6 kJ/molTautomerizationPEPPyruvate(unstable enol form)Pyruvate(stable keto)H2OC H2C COO–CH2C COO–OHCH3C COO–OO–OPO–O3.7 ● Complex Equilibria Involved in ATP HydrolysisSo far, as in Equation (3.33), the hydrolyses of ATP and other high-energy phos-phates have been portrayed as simple processes. The situation in a real bio-logical system is far more complex, owing to the operation of several ionic equi-libria. First, ATP, ADP, and the other species in Table 3.3 can exist in severaldifferent ionization states that must be accounted for in any quantitative analy-sis. Second, phosphate compounds bind a variety of divalent and monovalentcations with substantial affinity, and the various metal complexes must also beconsidered in such analyses. Consideration of these special cases makes thequantitative analysis far more realistic. The importance of these multiple equi-libria in group transfer reactions is illustrated for the hydrolysis of ATP, but theprinciples and methods presented are general and can be applied to any simi-lar hydrolysis reaction.The Multiple Ionization States of ATP and the pH Dependence of �G°�ATP has five dissociable protons, as indicated in Figure 3.15. Three of the pro-tons on the triphosphate chain dissociate at very low pH. The adenine ringamino group exhibits a pKa of 4.06, whereas the last proton to dissociate fromthe triphosphate chain possesses a pKa of 6.95. At higher pH values, ATP iscompletely deprotonated. ADP and phosphoric acid also undergo multiple ion-izations. These multiple ionizations make the equilibrium constant for ATPhydrolysis more complicated than the simple expression in Equation (3.35).Multiple ionizations must also be taken into account when the pH dependenceof �G° is considered. The calculations are beyond the scope of this text, butFigure 3.16 shows the variation of �G° as a function of pH. The free energyof hydrolysis is nearly constant from pH 4 to pH 6. At higher values of pH,�G° varies linearly with pH, becoming more negative by 5.7 kJ/mol for everypH unit of increase at 37°C. Because the pH of most biological tissues and flu-ids is near neutrality, the effect on �G ° is relatively small, but it must be takeninto account in certain situations.The Effect of Metal Ions on the Free Energy of Hydrolysis of ATPMost biological environments contain substantial amounts of divalent andmonovalent metal ions, including Mg2�, Ca2�, Na�, K�, and so on. What effectdo metal ions have on the equilibrium constant for ATP hydrolysis and the3.7 ● Complex Equilibria Involved in ATP Hydrolysis 77FIGURE 3.15 ● Adenosine 5�-triphosphate (ATP).FIGURE 3.16 ● The pH dependence of thefree energy of hydrolysis of ATP. Because pHvaries only slightly in biological environments,the effect on �G is usually small.–70∆G (kJ/mol)4pH–60–50–40–305 6 7 8 9 10 11 12 13–35.7OOOHPHO OP PO O OOH OHCH2 OHO OHNN NNNH3+Color indicates the locations of thefive dissociable protons of ATP.associated free energy change? Figure 3.17 shows the change in �G°� with pMg(that is, �log10[Mg2�]) at pH 7.0 and 38°C. The free energy of hydrolysis ofATP at zero Mg2� is �35.7 kJ/mol, and at 5 mM free Mg2� (the minimum inthe plot) the �Gobs° is approximately �31 kJ/mol. Thus, in most real biolog-ical environments (with pH near 7 and Mg2� concentrations of 5 mM or more)the free energy of hydrolysis of ATP is altered more by metal ions than by pro-tons. A widely used “consensus value” for �G°� of ATP in biological systems is�30.5 kJ/mol (Table 3.3). This value, cited in the 1976 Handbook of Biochemistryand Molecular Biology (3rd ed., Physical and Chemical Data, Vol. 1, pp. 296–304,Boca Raton, FL: CRC Press), was determined in the presence of “excess Mg2�.”This is the value we use for metabolic calculations in the balance of this text.The Effect of Concentration on the Free Energy of Hydrolysis of ATPThrough all these calculationsof the effect of pH and metal ions on the ATPhydrolysis equilibrium, we have assumed “standard conditions” with respect toconcentrations of all species except for protons. The levels of ATP, ADP, andother high-energy metabolites never even begin to approach the standard stateof 1 M. In most cells, the concentrations of these species are more typically 1to 5 mM or even less. Earlier, we described the effect of concentration on equi-librium constants and free energies in the form of Equation (3.12). For thepresent case, we can rewrite this as�G � �G° � RT ln (3.36)where the terms in brackets represent the sum () of the concentrations of allthe ionic forms of ATP, ADP, and Pi.It is clear that changes in the concentrations of these species can have largeeffects on �G. The concentrations of ATP, ADP, and Pi may, of course, varyrather independently in real biological environments, but if, for the sake ofsome model calculations, we assume that all three concentrations are equal,then the effect of concentration on �G is as shown in Figure 3.18. The freeenergy of hydrolysis of ATP, which is �35.7 kJ/mol at 1 M, becomes �49.4kJ/mol at 5 mM (that is, the concentration for which pC � �2.3 in Figure3.18). At 1 mM ATP, ADP, and Pi, the free energy change becomes even morenegative at �53.6 kJ/mol. Clearly, the effects of concentration are much greater thanthe effects of protons or metal ions under physiological conditions.Does the “concentration effect” change ATP’s position in the energy hier-archy (in Table 3.3)? Not really. All the other high- and low-energy phosphatesexperience roughly similar changes in concentration under physiological con-ditions and thus similar changes in their free energies of hydrolysis. The rolesof the very high-energy phosphates (PEP, 1,3-bisphosphoglycerate, and crea-tine phosphate) in the synthesis and maintenance of ATP in the cell are con-sidered in our discussions of metabolic pathways. In the meantime, several ofthe problems at the end of this chapter address some of the more interestingcases.3.8 ● The Daily Human Requirement for ATPWe can end this discussion of ATP and the other important high-energy com-pounds in biology by discussing the daily metabolic consumption of ATP byhumans. An approximate calculation gives a somewhat surprising and impres-sive result. Assume that the average adult human consumes approximately[ADP][Pi][ATP]78 Chapter 3 ● Thermodynamics of Biological SystemsFIGURE 3.17 ● The free energy of hydrolysisof ATP as a function of total Mg2� ion concen-tration at 38°C and pH 7.0. (Adapted from Gwynn, R. W., and Veech, R. L., 1973. The equilibrium constants of theadenosine triphosphate hydrolysis and the adenosine triphos-phate-citrate lyase reactions. Journal of Biological Chemistry248:6966–6972.)FIGURE 3.18 ● The free energy of hydrolysisof ATP as a function of concentration at 38°C,pH 7.0. The plot follows the relationshipdescribed in Equation (3.36), with the concen-trations [C] of ATP, ADP, and Pi assumed to beequal.1–36.0–35.0–34.0–33.0–32.0–31.0–30.0∆G°' (kJ/mol)2 3 4 5 6–Log10 [Mg]–35.70–40–45–50–55∆G (kJ/mol)1.0 2.0 3.0–Log10 [C]Where C = concentration of ATP, ADP, and Pi11,700 kJ (2800 kcal, that is, 2,800 Calories) per day. Assume also that the meta-bolic pathways leading to ATP synthesis operate at a thermodynamic efficiencyof approximately 50%. Thus, of the 11,700 kJ a person consumes as food, about5,860 kJ end up in the form of synthesized ATP. As indicated earlier, the hydrol-ysis of 1 mole of ATP yields approximately 50 kJ of free energy under cellularconditions. This means that the body cycles through 5860/50 � 117 moles ofATP each day. The disodium salt of ATP has a molecular weight of 551 g/mol,so that an average person hydrolyzes about(117 moles) � 64,467 g of ATP per dayThe average adult human, with a typical weight of 70 kg or so, thus consumesapproximately 65 kilograms of ATP per day, an amount nearly equal to his/herown body weight! Fortunately, we have a highly efficient recycling system forATP/ADP utilization. The energy released from food is stored transiently inthe form of ATP. Once ATP energy is used and ADP and phosphate are released,our bodies recycle it to ATP through intermediary metabolism, so that it maybe reused. The typical 70-kg body contains only about 50 grams of ATP/ADPtotal. Therefore, each ATP molecule in our bodies must be recycled nearly1300 times each day! Were it not for this fact, at current commercial prices ofabout $10 per gram, our ATP “habit” would cost approximately $650,000 perday! In these terms, the ability of biochemistry to sustain the marvelous activ-ity and vigor of organisms gains our respect and fascination.551 gmoleProblems 79PROBLEMS1. An enzymatic hydrolysis of fructose-1-P,Fructose-1-P � H2O 34 fructose � Piwas allowed to proceed to equilibrium at 25°C. The original con-centration of fructose-1-P was 0.2 M, but when the system hadreached equilibrium the concentration of fructose-1-P was only6.52 � 10�5 M. Calculate the equilibrium constant for this reac-tion and the free energy of hydrolysis of fructose-1-P.2. The equilibrium constant for some process A zy B is 0.5 at20°C and 10 at 30°C. Assuming that �H° is independent of tem-perature, calculate �H° for this reaction. Determine �G° and �S°at 20° and at 30°C. Why is it important in this problem to assumethat �H° is independent of temperature?3. The standard-state free energy of hydrolysis for acetyl phos-phate is �G ° � �42.3 kJ/mol.Acetyl-P � H2O 88n acetate � PiCalculate the free energy change for acetyl phosphate hydrolysisin a solution of 2 mM acetate, 2 mM phosphate, and 3 nM acetylphosphate.4. Define a state function. Name three thermodynamic quanti-ties that are state functions and three that are not.5. ATP hydrolysis at pH 7.0 is accompanied by release of a hydro-gen ion to the mediumATP4� � H2O 34 ADP3� � HPO42� � H�If the �G°� for this reaction is �30.5 kJ/mol, what is �G° (that is,the free energy change for the same reaction with all components,including H�, at a standard state of 1 M)?6. For the process A zy B, Keq(AB) is 0.02 at 37°C. For the processB zy C, Keq(BC) � 1000 at 37°C.a. Determine Keq(AC), the equilibrium constant for the overallprocess A zy C, from Keq(AB) and Keq(BC).b. Determine standard-state free energy changes for all threeprocesses, and use �G°(AC) to determine Keq(AC). Make surethat this value agrees with that determined in part a of this prob-lem.7. Draw all possible resonance structures for creatine phosphateand discuss their possible effects on resonance stabilization of themolecule.8. Write the equilibrium constant, Keq, for the hydrolysis of cre-atine phosphate and calculate a value for Keq at 25°C from thevalue of �G°� in Table 3.3.9. Imagine that creatine phosphate, rather than ATP, is the uni-versal energy carrier molecule in the human body. Repeat thecalculation presented in Section 3.8, calculating the weight ofcreatine phosphate that would need to be consumed each dayby a typical adult human if creatine phosphate could not be recy-cled. If recycling of creatine phosphate were possible, and if thetypical adult human body contained 20 grams of creatine phos-phate, how many times would each creatine phosphate moleculeneed to be turned over or recycled each day? Repeat the calcu-lation assuming that glycerol-3-phosphate is the universal energycarrier, and that the body contains 20 grams of glycerol-3-phos-phate.10. Calculate the free energy of hydrolysis of ATP in a rat livercell in which the ATP, ADP, and Pi concentrations are 3.4, 1.3, and4.8 mM, respectively.11. Hexokinase catalyzes the phosphorylation of glucose fromATP, yielding glucose-6-P and ADP. Using the values of Table 3.3,calculate the standard-state free energy change and equilibriumconstant for the hexokinase reaction.12. Would you expect the free energy of hydrolysis of aceto-acetyl-coenzyme A (see diagram) to be greater than, equal to, or lessthan that of acetyl-coenzyme A? Provide a chemical rationale foryour answer.13. Consider carbamoyl phosphate, a precursor in the biosynthe-sis of pyrimidines:Based on the discussion of high-energy phosphates in this chap-ter, would you expect carbamoyl phosphate to possess a high freeenergy of hydrolysis? Provide a chemical rationale for your answer.PO32–H3NCOO+CCH3 CH2O OC S CoA80 Chapter 3 ● Thermodynamics of Biological SystemsFURTHER READINGAlberty, R. A., 1968. Effect of pH and metal ion concentration on the equi-librium hydrolysis of adenosine triphosphate to adenosine diphosphate.Journal of Biological Chemistry 243:1337–1343.Alberty, R. A., 1969. Standard Gibbs free energy, enthalpy, and entropychanges as a function of pH and pMg for reactions involving adenosinephosphates. Journal of Biological Chemistry 244:3290–3302.Brandts, J. F., 1964. The thermodynamics of protein denaturation. I. Thedenaturation of chymotrypsinogen. Journal of the American Chemical Society86:4291–4301.Cantor, C. R., and Schimmel, P. R., 1980. Biophysical Chemistry. SanFrancisco: W.H. Freeman.Dickerson, R. E., 1969. Molecular Thermodynamics. New York: Benjamin Co.Edsall, J. T., and Gutfreund, H., 1983. Biothermodynamics: The Study ofBiochemical Processes at Equilibrium. New York: John Wiley.Edsall, J. T., and Wyman, J., 1958. Biophysical Chemistry. New York: AcademicPress.Gwynn, R. W., and Veech, R. L., 1973. The equilibrium constants of theadenosine triphosphate hydrolysis and the adenosine triphosphate-citratelyase reactions. Journal of Biological Chemistry 248:6966–6972.Klotz, I. M., 1967. Energy Changes in Biochemical Reactions. New York:Academic Press.Lehninger, A. L., 1972. Bioenergetics, 2nd ed. New York: Benjamin Co.Morris, J. G., 1968. A Biologist’s Physical Chemistry. Reading, MA: Addison-Wesley.Patton, A. R., 1965. Biochemical Energetics and Kinetics. Philadelphia: W.B.Saunders.Schrödinger, E., 1945. What Is Life? New York: Macmillan.Segel, I. H., 1976. Biochemical Calculations, 2nd ed. New York: John Wiley.Tanford, C., 1980. The Hydrophobic Effect, 2nd ed. New York: John Wiley.81Chapter 4Amino AcidsAll objects have mirror images. Like many biomolecules, amino acids exist in mirror-imageforms (stereoisomers) that are not superimposable. Only the L-isomers of amino acids commonlyoccur in nature. ( The Mirror of Venus (1898), Sir Edward Burne-Jones/Museu Calouste Gulbenkian Lisbon/TheBridgeman Art Library)Proteins are the indispensable agents of biological function, and amino acidsare the building blocks of proteins. The stunning diversity of the thousands ofproteins found in nature arises from the intrinsic properties of only 20 com-monly occurring amino acids. These features include (1) the capacity to poly-merize, (2) novel acid–base properties, (3) varied structure and chemical func-tionality in the amino acid side chains, and (4) chirality. This chapter describeseach of these properties, laying a foundation for discussions of protein struc-ture (Chapters 5 and 6), enzyme function (Chapters 14–16), and many othersubjects in later chapters.To hold, as ’twere, the mirror up to nature.WILLIAM SHAKESPEARE, HamletOUTLINE4.1 ● Amino Acids: Building Blocks ofProteins4.2 ● Acid–Base Chemistry of Amino Acids4.3 ● Reactions of Amino Acids4.4 ● Optical Activity and Stereochemistry ofAmino Acids4.5 ● Spectroscopic Properties of AminoAcids4.6 ● Separation and Analysis of Amino AcidMixtures4.1 ● Amino Acids: Building Blocks of ProteinsStructure of a Typical Amino AcidThe structure of a single typical amino acid is shown in Figure 4.1. Central tothis structure is the tetrahedral alpha (�) carbon (C�), which is covalentlylinked to both the amino group and the carboxyl group. Also bonded to this�-carbon is a hydrogen and a variable side chain. It is the side chain, the so-called R group, that gives each amino acid its identity. The detailed acid–baseproperties of amino acids are discussed in the following sections. It is sufficientfor now to realize that, in neutral solution (pH 7), the carboxyl group existsas OCOO� and the amino group as ONH3�. Because the resulting amino acidcontains one positive and one negative charge, it is a neutral molecule calleda zwitterion. Amino acids are also chiral molecules. With four different groupsattached to it, the �-carbon is said to be asymmetric. The two possible configu-rations for the �-carbon constitute nonidentical mirror image isomers or enan-tiomers. Details of amino acid stereochemistry are discussed in Section 4.4.Amino Acids Can Join via Peptide BondsThe crucial feature of amino acids that allows them to polymerize to form pep-tides and proteins is the existence of their two identifying chemical groups: theamino (ONH3�) and carboxyl (OCOO�) groups, as shown in Figure 4.2. Theamino and carboxyl groups of amino acids can react in a head-to-tail fashion,eliminating a water molecule and forming a covalent amide linkage, which, inthe case of peptides and proteins, is typically referred to as a peptide bond.The equilibrium for this reaction in aqueous solution favors peptide bondhydrolysis. For this reason, biological systems as well as peptide chemists in thelaboratory must carry out peptide bond formation in an indirect manner orwith energy input.Iteration of the reaction shown in Figure 4.2 produces polypeptides andproteins. The remarkable properties of proteins, which we shall discover andcome to appreciate in later chapters, all depend in one way or another on theunique properties and chemical diversity of the 20 common amino acids foundin proteins.Common Amino AcidsThe structures and abbreviations for the 20 amino acids commonly found inproteins are shown in Figure 4.3. All the amino acids except proline have bothfree �-amino and free �-carboxyl groups (Figure 4.1). There are several waysto classify the common amino acids. The most useful of these classifications isbased on the polarity of the side chains. Thus, the structures shown in Figure4.3 are grouped into the following categories: (1) nonpolar or hydrophobic82 Chapter 4 ● Amino AcidsFIGURE 4.1 ● Anatomy of an amino acid. Except for proline and its derivatives, all ofthe amino acids commonly found in proteins possess this type of structure.H RH3N COO–Cαα-CarbonSidechainAminogroupCarboxylgroupBall-and-stickmodelAmino acids aretetrahedral structuresRCOO–NH3 NH3RCOO–+amino acids, (2) neutral (uncharged) but polar amino acids, (3) acidic aminoacids (which have a net negative charge at pH 7.0), and (4) basic amino acids(which have a net positive charge at neutral pH). In later chapters, the impor-tance of this classification system for predicting protein properties becomesclear. Also shown in Figure 4.3 are the three-letter and one-letter codes usedto represent the amino acids. These codes are useful when displaying and com-paring the sequences of proteins in shorthand form. (Note that several of theone-letter abbreviations are phonetic in origin: arginine � “Rginine” � R,phenylalanine � “Fenylalanine” � F, aspartic acid � “asparDic” � D.)Nonpolar Amino AcidsThe nonpolar amino acids (Figure 4.3a) include all those with alkyl chain Rgroups (alanine, valine, leucine, and isoleucine), as well as proline (with itsunusual cyclicstructure), methionine (one of the two sulfur-containing aminoacids), and two aromatic amino acids, phenylalanine and tryptophan.Tryptophan is sometimes considered a borderline member of this groupbecause it can interact favorably with water via the N–H moiety of the indolering. Proline, strictly speaking, is not an amino acid but rather an �-imino acid.Polar, Uncharged Amino AcidsThe polar, uncharged amino acids (Figure 4.3b) except for glycine contain Rgroups that can form hydrogen bonds with water. Thus, these amino acids areusually more soluble in water than the nonpolar amino acids. Several excep-tions should be noted. Tyrosine displays the lowest solubility in water of the 20common amino acids (0.453 g/L at 25°C). Also, proline is very soluble in water,and alanine and valine are about as soluble as arginine and serine. The amidegroups of asparagine and glutamine; the hydroxyl groups of tyrosine, threo-nine, and serine; and the sulfhydryl group of cysteine are all good hydrogen4.1 ● Amino Acids: Building Blocks of Proteins 83(Text continues on page 86.)FIGURE 4.2 ● The �-COOH and �-NH3�groups of two amino acids can react with theresulting loss of a water molecule to form acovalent amide bond. (Irving Geis.)– –++Removal of a water molecule......formation of the CO—NH Amino endPeptide bondCarboxyl end+–+–Two amino acidsRNOCOCαNOCCα–H2OHHHHO+84H3N+COOHPhenylalanine (Phe, F)C H CH2Methionine (Met, M)H3N+COOHC H CH2CH2SCH3Tryptophan (Trp, W)H3N+COOHC H CH2CNHH3N+COOHHistidine (His, H)H3N+COOHTyrosine (Tyr, Y)C H C H CCH2CH2H3N+COOHArginine (Arg, R)Lysine (Lys, K)C H (d)—BasicCH2OHHCNHH+NCHCH2CH2CH2NH3+H3N+COOHCCH2CH2CH2NHCH NH2H2+NH3N+COOHIsoleucine (Ile, I)C H CH2H3CCH3CH3N+COOHThreonine (Thr, T)H3N+COOHCysteine (Cys, C)C H C H CH3CH2SHH C OH H CHFIGURE 4.3 ● The 20 amino acids that are the building blocks of most proteins can be classified as (a) nonpolar (hydrophobic), (b) polar, neutral, (c) acidic, or (d) basic.85Proline (Pro, P)CCOOHHCH2CH2H2CH2 NGlutamine (Gln, Q)CH2CH3N+COOHH OCNH2CH2CH3H3N+COOHAlanine (Ala, A)C H Valine (Val, V)Leucine (Leu, L)H3C CH3CHH3N+COOHC H CH2H3N+COOHSerine (Ser, S)H3N+COOHGlycine (Gly, G)C H C H (b)—Polar, unchargedCH2OHHH3N+COOH H3N+COOHGlutamic acid (Glu, E)Aspartic acid (Asp, D)C H C H (c)—AcidicCH2COOHCH2COOHCH2Asparagine (Asn, N)OCNH2CH2CH3N+COOHH CH3CHH3N+COOHC H CH3+(a) Nonpolar (hydrophobic) Also shown are the one-letter and three-letter codes used to denote amino acids. For each amino acid, theball-and-stick (left) and space-filling (right) models show only the side chain. (Irving Geis)bond–forming moieties. Glycine, the simplest amino acid, has only a singlehydrogen for an R group, and this hydrogen is not a good hydrogen bond for-mer. Glycine’s solubility properties are mainly influenced by its polar aminoand carboxyl groups, and thus glycine is best considered a member of the polar,uncharged group. It should be noted that tyrosine has significant nonpolarcharacteristics due to its aromatic ring and could arguably be placed in thenonpolar group (Figure 4.3a). However, with a pKa of 10.1, tyrosine’s pheno-lic hydroxyl is a charged, polar entity at high pH.Acidic Amino AcidsThere are two acidic amino acids—aspartic acid and glutamic acid—whose Rgroups contain a carboxyl group (Figure 4.3c). These side chain carboxylgroups are weaker acids than the �-COOH group, but are sufficiently acidic toexist as OCOO� at neutral pH. Aspartic acid and glutamic acid thus have anet negative charge at pH 7. These negatively charged amino acids play sev-eral important roles in proteins. Many proteins that bind metal ions for struc-tural or functional purposes possess metal binding sites containing one or moreaspartate and glutamate side chains. Carboxyl groups may also act as nucleo-philes in certain enzyme reactions and may participate in a variety of elec-trostatic bonding interactions. The acid–base chemistry of such groups is con-sidered in detail in Section 4.2.Basic Amino AcidsThree of the common amino acids have side chains with net positive chargesat neutral pH: histidine, arginine, and lysine (Figure 4.3d). The ionized groupof histidine is an imidazolium, that of arginine is a guanidinium, and lysinecontains a protonated alkyl amino group. The side chains of the latter twoamino acids are fully protonated at pH 7, but histidine, with a side chain pKaof 6.0, is only 10% protonated at pH 7. With a pKa near neutrality, histidineside chains play important roles as proton donors and acceptors in manyenzyme reactions. Histidine-containing peptides are important biologicalbuffers, as discussed in Chapter 2. Arginine and lysine side chains, which areprotonated under physiological conditions, participate in electrostatic interac-tions in proteins.Uncommon Amino AcidsSeveral amino acids occur only rarely in proteins (Figure 4.4). These includehydroxylysine and hydroxyproline, which are found mainly in the collagen andgelatin proteins, and thyroxine and 3,3�,5-triiodothyronine, iodinated aminoacids that are found only in thyroglobulin, a protein produced by the thyroidgland. (Thyroxine and 3,3�,5-triiodothyronine are produced by iodination oftyrosine residues in thyroglobulin in the thyroid gland. Degradation of thy-roglobulin releases these two iodinated amino acids, which act as hormones to regulate growth and development.) Certain muscle proteins contain methy-lated amino acids, including methylhistidine, � -N -methyllysine, and � -N,N,N -trimethyllysine (Figure 4.4). �-Carboxyglutamic acid is found in several pro-teins involved in blood clotting, and pyroglutamic acid is found in a uniquelight-driven proton-pumping protein called bacteriorhodopsin, which is dis-cussed elsewhere in this book. Certain proteins involved in cell growth andregulation are reversibly phosphorylated on the OOH groups of serine, thre-onine, and tyrosine residues. Aminoadipic acid is found in proteins isolatedfrom corn. Finally, N -methylarginine and N -acetyllysine are found in histoneproteins associated with chromosomes.86 Chapter 4 ● Amino AcidsAmino Acids Not Found in ProteinsCertain amino acids and their derivatives, although not found in proteins,nonetheless are biochemically important. A few of the more notable examplesare shown in Figure 4.5. �-Aminobutyric acid, or GABA, is produced by thedecarboxylation of glutamic acid and is a potent neurotransmitter. Histamine,which is synthesized by decarboxylation of histidine, and serotonin, which isderived from tryptophan, similarly function as neurotransmitters and regula-tors. �-Alanine is found in nature in the peptides carnosine and anserine andis a component of pantothenic acid (a vitamin), which is a part of coenzymeA. Epinephrine (also known as adrenaline), derived from tyrosine, is an impor-tant hormone. Penicillamine is a constituent of the penicillin antibiotics.Ornithine, betaine, homocysteine, and homoserine are important metabolicintermediates. Citrulline is the immediate precursor of arginine.4.1 ● Amino Acids: Building Blocks of Proteins 87FIGURE 4.4 ● The structures of several amino acids that are less commonbut neverthe-less found in certain proteins. Hydroxylysine and hydroxyproline are found in connective-tissue proteins, pyroglutamic acid is found in bacteriorhodopsin (a protein inHalobacterium halobium), and aminoadipic acid is found in proteins isolated from corn.COOHCH3N HCH2NHH2N +NHCH3N-MethylarginineCH2CH2CCOOHCH3N HCH2N-AcetyllysineCH2CH2NHCH2C CH3OCOOHCH3N HCH25-HydroxylysineCH2CH OHCH2NH3+CCH2H2CHN C HCOOHH OH4-HydroxyprolineCOOHCH3N HCH2ThyroxineI IOI IOHCOOHCH3N HCH23-MethylhistidineCCHNHH3CCNCOOHCH3N HCH2ε-N-MethyllysineCH2CH2CH2NH2+CH3COOHCH3N HCH2ε-N,N,N-Trimethyl-lysineCH2CH2CH2N+(CH3)3COOHCH3N HCH2Aminoadipic acidCH2CH2COOHCOOHCH3N HCH2γ-Carboxyglutamic acidCHCOOHHOOCC H2CH2CHN C HCOOHPyroglutamic acidOCOOHCH3N HCH2PhosphoserineOPO3H2COOHCH3N HCPhosphothreonineCH3HCOOHCH3N HCH2Phosphotyrosine+ ++ + + + ++ + + + +OPO3H2OPO3H2+H4.2 ● Acid–Base Chemistry of Amino AcidsAmino Acids Are Weak Polyprotic AcidsFrom a chemical point of view, the common amino acids are all weak polypro-tic acids. The ionizable groups are not strongly dissociating ones, and thedegree of dissociation thus depends on the pH of the medium. All the aminoacids contain at least two dissociable hydrogens.Consider the acid–base behavior of glycine, the simplest amino acid. Atlow pH, both the amino and carboxyl groups are protonated and the moleculehas a net positive charge. If the counterion in solution is a chloride ion, thisform is referred to as glycine hydrochloride. If the pH is increased, the car-boxyl group is the first to dissociate, yielding the neutral zwitterionic speciesGly0 (Figure 4.6). Further increase in pH eventually results in dissociation ofthe amino group to yield the negatively charged glycinate. If we denote these88 Chapter 4 ● Amino AcidsHCCl2ONHCHH2COC CHC NH3H2N+ CH2COOHSarcosine(N-methylglycine)H3C(CH2)3COOHNH3+γ-Aminobutyric acid(GABA)N+ CH2COOHBetaine(N,N,N-trimethylglycine)CH3H3CH3C CH2NH3+β-AlanineCH2COOHCH2OAzaserineO-diazoacetylserineCHCOOHH3N+N+ N–H3N+ CHCOOHHomoserineCH2CH2OHH3N+ CHCOOHL-LanthionineCH2 SHCCOOHCH2NH3+ H3N+ CHCOOHHomocysteineCH2CH2SHH3N+ CHCOOHCHOHL-PhenylserineHN CHCH2OHCHOHL-ChloramphenicolNO2OOCycloserineCH2NH3+HistamineCH2NHNHONHCH2CH2 NH3+SerotoninH3N CCOOH+HH3N C CH3SHPenicillamineH3N CCOOH+HCH2CH2CH2N HC ONH2CitrullineH3N CCOOH+HCH2CH2CH2NH3+OrnithineHONH2+CH3CH2C HOHOHEpinephrine+FIGURE 4.5 ● The structures of some amino acids that are not normally found in pro-teins but that perform other important biological functions. Epinephrine, histamine, andserotonin, although not amino acids, are derived from and closely related to amino acids.three forms as Gly�, Gly0, and Gly�, we can write the first dissociation of Gly�asGly� � H2O 34 Gly0 � H3O�and the dissociation constant K1 asValues for K1 for the common amino acids are typically 0.4 to 1.0 � 10�2 M,so that typical values of pK1 center on values of 2.0 to 2.4 (see Table 4.1). Ina similar manner, we can write the second dissociation reaction asGly0 � H2O 34 Gly� � H3O�K1 �[Gly0][H3O�][Gly �]4.2 ● Acid–Base Chemistry of Amino Acids 89Table 4.1pKa Values of Common Amino AcidsAmino Acid �-COOH pK a �-NH3� pK a R group pK aAlanine 2.4 9.7Arginine 2.2 9.0 12.5Asparagine 2.0 8.8Aspartic acid 2.1 9.8 3.9Cysteine 1.7 10.8 8.3Glutamic acid 2.2 9.7 4.3Glutamine 2.2 9.1Glycine 2.3 9.6Histidine 1.8 9.2 6.0Isoleucine 2.4 9.7Leucine 2.4 9.6Lysine 2.2 9.0 10.5Methionine 2.3 9.2Phenylalanine 1.8 9.1Proline 2.1 10.6Serine 2.2 9.2 �13.00Threonine 2.6 10.4 �13.00Tryptophan 2.4 9.4Tyrosine 2.2 9.1 10.1Valine 2.3 9.6FIGURE 4.6 ● The ionic forms of the aminoacids, shown without consideration of any ion-izations on the side chain. The cationic form isthe low pH form, and the titration of thecationic species with base yields the zwitterionand finally the anionic form. (Irving Geis)H3N C HCOOHR+R+NOCOpH 1 Net charge +1 pH 7 Net charge 0 pH 13 Net charge –1CαROCO–+NCαROO–NCationic formH3N C HCOO–RH2N C HCOO–RZwitterion (neutral) Anionic form+CαCH+ H+and the dissociation constant K2 asTypical values for pK2 are in the range of 9.0 to 9.8. At physiological pH, the�-carboxyl group of a simple amino acid (with no ionizable side chains) is com-pletely dissociated, whereas the �-amino group has not really begun its disso-ciation. The titration curve for such an amino acid is shown in Figure 4.7.EXAMPLEWhat is the pH of a glycine solution in which the �-NH3� group is one-thirddissociated?SOLUTIONThe appropriate Henderson–Hasselbalch equation isIf the �-amino group is one-third dissociated, there is one part Gly� for everytwo parts Gly0. The important pKa is the pKa for the amino group. The glycine�-amino group has a pKa of 9.6. The result ispH � 9.6 � log10 (1/2)pH � 9.3Note that the dissociation constants of both the �-carboxyl and �-aminogroups are affected by the presence of the other group. The adjacent �-aminogroup makes the �-COOH group more acidic (that is, it lowers the pKa) sopH � pK a � log10[Gly�][Gly0]K2 �[Gly�][H3O�][Gly0]90 Chapter 4 ● Amino AcidsFIGURE 4.7 ● Titration of glycine, a simple amino acid. The isoelectric point, pI, thepH where the molecule has a net charge of 0, is defined as (pK1 � pK2)/2.024681012141.0 0 1.0Equivalents of H+ pK2IsoelectricpointpK1CH2H3N+COOHCH2H3N+COO–CH2H2NCOO–Gly+ Gly0 Gly–0 1.0 2.0Equivalents of OH– addedEquivalents of OH–2.0 0Equivalents of H+ added1.0pHthat it gives up a proton more readily than simple alkyl carboxylic acids. Thus,the pK1 of 2.0 to 2.1 for �-carboxyl groups of amino acids is substantially lowerthan that of acetic acid (pKa � 4.76), for example. What is the chemical basisfor the low pKa of the �-COOH group of amino acids? The �-NH3� (ammo-nium) group is strongly electron-withdrawing, and the positive charge of theamino group exerts a strong field effect and stabilizes the carboxylate anion.(The effect of the �-COO� group on the pKa of the �-NH3� group is the basisfor Problem 4 at the end of this chapter.)Ionization of Side ChainsAs we have seen, the side chains of several of the amino acids also contain dis-sociable groups. Thus, aspartic and glutamic acids contain an additional car-boxyl function, and lysine possesses an aliphatic amino function. Histidine con-tains an ionizable imidazolium proton, and arginine carries a guanidiniumfunction. Typical pKa values of these groups are shown in Table 4.1. The �-car-boxyl group of aspartic acid and the �-carboxyl side chain of glutamic acidexhibit pKa values intermediate to the �-COOH on the one hand and typicalaliphatic carboxyl groupson the other hand. In a similar fashion, the �-amino group of lysine exhibits a pKa that is higher than the �-amino groupbut similar to that for a typical aliphatic amino group. These intermediate valuesfor side-chain pKa values reflect the slightly diminished effect of the �-carbondissociable groups that lie several carbons removed from the side-chain func-tional groups. Figure 4.8 shows typical titration curves for glutamic acid andlysine, along with the ionic species that predominate at various points in the4.2 ● Acid–Base Chemistry of Amino Acids 910 1.0 2.0 3.0Equivalents of OH– added0 1.0 2.0 3.0Equivalents of OH– added02468101214pK3pK2pK1 IsoelectricpointCOOH COO– COO–02468101214COO– COO– COO–Isoelectric pointH3N+COOHC H CH2COOHCH2H3N+COO–C H CH2CH2H3N+COO–C H CH2CH2H2NCOO–C H CH2CH2NH3+H3N+ C H CH2CH2CH2CH2NH3H3N+ C H CH2CH2CH2CH2NH3+H2N C H CH2CH2CH2CH2NH3+H2N C H CH2CH2CH2CH2NH2COOHpK3pK2pK1Glu+ Glu0 Glu– Glu2–Lys2+ Lys+ Lys0 Lys–pH pHFIGURE 4.8 ● Titrations of glutamic acid and lysine.92 Chapter 4 ● Amino AcidsC R I T I C A L D E V E L O P M E N T S I N B I O C H E M I S T R YGreen Fluorescent Protein—The “Light Fantastic” from Jellyfish to Gene ExpressionAquorea victoria, a species of jellyfish found in the northwest PacificOcean, contains a green fluorescent protein (GFP) that workstogether with another protein, aequorin, to provide a defensemechanism for the jellyfish. When the jellyfish is attacked orshaken, aequorin produces a blue light. This light energy is cap-tured by GFP, which then emits a bright green flash that pre-sumably blinds or startles the attacker. Remarkably, the fluores-cence of GFP occurs without the assistance of a prosthetic group—a “helper molecule” that would mediate GFP’s fluorescence.Instead, the light-transducing capability of GFP is the result of areaction between three amino acids in the protein itself. As shownbelow, adjacent serine, tyrosine, and glycine in the sequence ofthe protein react to form the pigment complex—termed a chro-mophore. No enzymes are required; the reaction is autocatalytic.Because the light-transducing talents of GFP depend only onthe protein itself (upper photo, chromophore highlighted), GFPhas quickly become a darling of genetic engineering laboratories.The promoter of any gene whose cellular expression is of inter-est can be fused to the DNA sequence coding for GFP. Telltalegreen fluorescence tells the researcher when this fused gene hasbeen expressed (see lower photo and also Chapter 13).Boxer, S.G., 1997. Another green revolution. Nature 383:484–485.Autocatalytic oxidation of GFP amino acids leads to the chromophoreshown on the left. The green fluorescence requires further interactionsof the chromophore with other parts of the protein.OHOHHOO2Phe-Ser-Tyr-Gly-Val-Gln64 69 NNNPheHGlnValOtitration. The only other side-chain groups that exhibit any significant degreeof dissociation are the para-OH group of tyrosine and the OSH group of cys-teine. The pKa of the cysteine sulfhydryl is 8.32, so that it is about 12% disso-ciated at pH 7. The tyrosine para-OH group is a very weakly acidic group, witha pKa of about 10.1. This group is essentially fully protonated and unchargedat pH 7.4.3 ● Reactions of Amino AcidsCarboxyl and Amino Group ReactionsThe �-carboxyl and �-amino groups of all amino acids exhibit similar chemi-cal reactivity. The side chains, however, exhibit specific chemical reactivities,depending on the nature of the functional groups. Whereas all of these reac-tivities are important in the study and analysis of isolated amino acids, it is thecharacteristic behavior of the side chain that governs the reactivity of aminoacids incorporated into proteins. There are three reasons to consider thesereactivities. Proteins can be chemically modified in very specific ways by takingadvantage of the chemical reactivity of certain amino acid side chains. Thedetection and quantification of amino acids and proteins often depend on reac-tions that are specific to one or more amino acids and that result in color,radioactivity, or some other quantity that can be easily measured. Finally andmost importantly, the biological functions of proteins depend on the behaviorand reactivity of specific R groups.The carboxyl groups of amino acids undergo all the simple reactions com-mon to this functional group. Reaction with ammonia and primary aminesyields unsubstituted and substituted amides, respectively (Figure 4.9a,b). Esters4.3 ● Reactions of Amino Acids 93FIGURE 4.9 ● Typical reactions of the com-mon amino acids (see text for details).+C COOHRNH3+Amino acidNH3H2OC CRH3N+ONH2Amide+ NH2H2ONHSubstituted amideR' R'+ OHH2OEsterOR'+ NH2CHRCOR'OHNHCHRC NHCHRCOPolymerNHCHR COR'+CHRNH3+Amino acidH2OC NSchiff baseAMINO GROUP REACTIONSHR'+ CHClHNSubstituted amideOR'ClCARBOXYL GROUP REACTIONSH HC CRH3N+OHR' C CRH3N+OHOCCOO–CHRCOO–COCCHRCOO–CC(a)(b)(c)(d)(e)(f)C HCR'OCR'O++H+H+Amino acidAmino acidAmino acidand acid chlorides are also readily formed. Esterification proceeds in the pres-ence of the appropriate alcohol and a strong acid (Figure 4.9c). Polymerizationcan occur by repetition of the reaction shown in Figure 4.9d. Free amino groupsmay react with aldehydes to form Schiff bases (Figure 4.9e) and can be acy-lated with acid anhydrides and acid halides (Figure 4.9f).The Ninhydrin ReactionAmino acids can be readily detected and quantified by reaction with ninhy-drin. As shown in Figure 4.10, ninhydrin, or triketohydrindene hydrate, is astrong oxidizing agent and causes the oxidative deamination of the �-aminofunction. The products of the reaction are the resulting aldehyde, ammonia,carbon dioxide, and hydrindantin, a reduced derivative of ninhydrin. Theammonia produced in this way can react with the hydrindantin and anothermolecule of ninhydrin to yield a purple product (Ruhemann’s Purple) thatcan be quantified spectrophotometrically at 570 nm. The appearance of CO2can also be monitored. Indeed, CO2 evolution is diagnostic of the presence ofan �-amino acid. �-Imino acids, such as proline and hydroxyproline, give brightyellow ninhydrin products with absorption maxima at 440 nm, allowing theseto be distinguished from the �-amino acids. Because amino acids are one ofthe components of human skin secretions, the ninhydrin reaction was onceused extensively by law enforcement and forensic personnel for fingerprintdetection. (Fingerprints as old as 15 years can be successfully identified usingthe ninhydrin reaction.) More sensitive fluorescent reagents are now used rou-tinely for this purpose.Specific Reactions of Amino Acid Side ChainsA number of reactions of amino acids have become important in recent yearsbecause they are essential to the degradation, sequencing, and chemical syn-thesis of peptides and proteins. These reactions are discussed in Chapter 5.94 Chapter 4 ● Amino AcidsFIGURE 4.10 ● The pathway of the ninhy-drin reaction, which produces a colored prod-uct called “Ruhemann’s Purple” that absorbslight at 570 nm. Note that the reaction involvesand consumes two molecules of ninhydrin.OOHC+OOHNinhydrinH3+N HCOOHR+ RCHO + CO2 + NH3 +NHHydrindantinHOHOHOHOOHO ON–OOOOOOOOTwo resonanceforms ofRuhemann’sPurple+ H+2nd NinhydrinIn recent years, biochemists have developed an arsenal of reactions thatare relatively specific to the side chains of particular amino acids. These reac-tions can be used to identify functional amino acids at the active sites of enzymesor to label proteins with appropriate reagents for further study. Cysteineresidues in proteins, for example, react with one another to form disulfidespecies and also react with a number of reagents, including maleimides (typi-cally N-ethylmaleimide), as shown in Figure 4.11. Cysteines also react effectively4.3 ● Reactions of Amino Acids 95FIGURE 4.11 ● Reactions of amino acid side-chain functional groups.LYSINEO2N S–OOCS NO2COO–5,5'–Dithiobis (2-nitrobenzoic acid)DTNB“Ellman’s reagent”S NO2COO–+ –S NO2COO–Thiol anion(λmax = 412 nm)C NCHH2CAcrylonitrileICH2COO–Iodoacetate+HO Hg COOHp–Hydroxy–mercuribenzoateH3+NC–OOC CH2 CH2CH2CH2 NH3+ CR'Schiff baseLysine+R' COHR groupHH3+NC–OOC CH2 CH2CH2CH2 NHHH3+NC–OOC CH2HSHH3+NC–OOC CH2HHg COOH+H3+NC–OOC CH2HSHH3+NC–OOC CH2HSH3+NC–OOC CH2HSH+H3+NC–OOC CH2HS CH2 CH2 C NH3+NC–OOC CH2HSH+H3+NC–OOC CH2HS CH2 COO– HI+S H2O+H2O+ H++OON CH2CH3N-EthylmaleimideOONHCYSTEINER groupC–OOC CH2HSHH3+NCystineCysteineNH3+CCH2 COO–HH3+NC–OOC CH2HSH3+NC–OOC CH2HSH S+H3+NC–OOC CH2HSCH2CH3HH2with iodoacetic acid to yield S-carboxymethyl cysteine derivatives. There arenumerous other reactions involving specialized reagents specific for particularside chain functional groups. Figure 4.11 presents a representative list of thesereagents and the products that result. It is important to realize that few if anyof these reactions are truly specific for one functional group; consequently,care must be exercised in their use.4.4 ● Optical Activity and Stereochemistry of Amino AcidsAmino Acids Are Chiral MoleculesExcept for glycine, all of the amino acids isolated from proteins have four dif-ferent groups attached to the �-carbon atom. In such a case, the �-carbon issaid to be asymmetric or chiral (from the Greek cheir, meaning “hand”), andthe two possible configurations for the �-carbon constitute nonsuperimposablemirror image isomers, or enantiomers (Figure 4.12). Enantiomeric moleculesdisplay a special property called optical activity—the ability to rotate the planeof polarization of plane-polarized light. Clockwise rotation of incident light isreferred to as dextrorotatory behavior, and counterclockwise rotation is calledlevorotatory behavior. The magnitude and direction of the optical rotationdepend on the nature of the amino acid side chain. The temperature, the wave-length of the light used in the measurement, the ionization state of the aminoacid, and therefore the pH of the solution, can also affect optical rotationbehavior. As shown in Table 4.2, some protein-derived amino acids at a givenpH are dextrorotatory and others are levorotatory, even though all of themare of the L configuration. The direction of optical rotation can be specifiedin the name by using a (�) for dextrorotatory compounds and a (�) for lev-orotatory compounds, as in L(�)-leucine.Nomenclature for Chiral MoleculesThe discoveries of optical activity and enantiomeric structures (see the box,page 97) made it important to develop suitable nomenclature for chiral mole-cules. Two systems are in common use today: the so-called D,L system and the(R,S) system.In the D,L system of nomenclature, the (�) and (�) isomers of glycer-aldehyde are denoted as D-glyceraldehyde and L-glyceraldehyde, respectively(Figure 4.13). Absolute configurations of all other carbon-based molecules arereferenced to D- and L-glyceraldehyde. When sufficient care is taken to avoidracemization of the amino acids during hydrolysis of proteins, it is found thatall of the amino acids derived from natural proteins are of the L configuration.Amino acids of the D configuration are nonetheless found in nature, especiallyas components of certain peptide antibiotics, such as valinomycin, gramicidin,and actinomycin D, and in the cell walls of certain microorganisms.In spite of its widespread acceptance, problems exist with the D,L systemof nomenclature. For example, this system can be ambiguous for moleculeswith two or more chiral centers. To address such problems, the (R,S ) systemof nomenclature for chiral molecules was proposed in 1956 by Robert Cahn,Sir Christopher Ingold, and Vladimir Prelog. In this more versatile system, pri-orities are assigned to each of the groups attached to a chiral center on thebasis of atomic number, atoms with higher atomic numbers having higher pri-orities (see the box, page 100).The newer (R,S) system of nomenclature is superior to the older D,L sys-tem in one important way. The configuration of molecules with more than one96 Chapter 4 ● Amino AcidsTable 4.2Specific Rotations for Some Amino AcidsSpecific Rotation [�]D25,Amino Acid DegreesL-Alanine �1.8L-Arginine �12.5L-Aspartic acid �5.0L-Glutamic acid �12.0L-Histidine �38.5L-Isoleucine �12.4L-Leucine �11.0L-Lysine �13.5L-Methionine �10.0L-Phenylalanine �34.5L-Proline �86.2L-Serine �7.5L-Threonine �28.5L-Tryptophan �33.7L-Valine �5.6FIGURE 4.12 ● Enantiomeric moleculesbased on a chiral carbon atom. Enantiomersare nonsuperimposable mirror images of eachother.C CWYWW WY YYX XZX Z Z XZPerspective drawingFischer projectionschiral center can be more easily, completely, and unambiguously described with(R,S) notation. Several amino acids, including isoleucine, threonine, hydroxy-proline, and hydroxylysine, have two chiral centers. In the (R,S) system, L-thre-onine is (2S,3R )-threonine. A chemical compound with n chiral centers canexist in 2n-isomeric structures, and the four amino acids just listed can thus eachtake on four different isomeric configurations. This amounts to two pairs ofenantiomers. Isomers that differ in configuration at only one of the asymmet-ric centers are non–mirror image isomers or diastereomers. The four stereo-4.4 ● Optical Activity and Stereochemistry of Amino Acids 97C R I T I C A L D E V E L O P M E N T S I N B I O C H E M I S T R YDiscovery of Optically Active Molecules and Determination of Absolute ConfigurationThe optical activity of quartz and certain other materials was firstdiscovered by Jean-Baptiste Biot in 1815 in France, and in 1848a young chemist in Paris named Louis Pasteur made a related andremarkable discovery. Pasteur noticed that preparations of opti-cally inactive sodium ammonium tartrate contained two visiblydifferent kinds of crystals that were mirror images of each other.Pasteur carefully separated the two types of crystals, dissolvedthem each in water, and found that each solution was opticallyactive. Even more intriguing, the specific rotations of these twosolutions were equal in magnitude and of opposite sign. Becausethese differences in optical rotation were apparent properties ofthe dissolved molecules, Pasteur eventually proposed that the mole-cules themselves were mirror images of each other, just like theirrespective crystals. Based on this and other related evidence, in1847 van’t Hoff and LeBel proposed the tetrahedral arrangementof valence bonds to carbon.In 1888, Emil Fischer decided that it should be possible todetermine the relative configuration of (�)-glucose, a six-carbonsugar with four asymmetric centers (see figure). Because each ofthe four C could be either of two configurations, glucose con-ceivably could exist in any one of 16 possible isomeric structures.It took three years to complete the solution of an elaborate chem-ical and logical puzzle. By 1891, Fischer had reduced his puzzleto a choice between two enantiomeric structures. (Methods fordetermining absolute configuration were not yet available, soFischer made a simple guess, selecting the structure shown in thefigure.) For this remarkable feat, Fischer received the Nobel Prizein chemistry in 1902. Sadly, Fischer, a brilliant but troubledchemist, later committed suicide.The absolute choice between Fischer’s two enantiomeric pos-sibilities would not be made for a long time. In 1951, J.M. Bijvoetin Utrecht, the Netherlands, used a new X-ray diffraction tech-nique to determine the absolute configuration of (among otherthings) the sodium rubidium salt of (�)-tartaric acid. Because thetartaric acid configuration could be related to that of glyceralde-hyde and because sugar and amino acid configurations could allbe related to glyceraldehyde, it became possible to determine theabsolute configuration of sugars and the common amino acids.The absolute configuration of tartaric acid determined by Bijvoetturned out to be the configuration that, up to then, had onlybeen assumed. This meant that Emil Fischer’s arbitrary guess 60years earlier had been correct.It was M.A. Rosanoff, a chemist and instructor at New YorkUniversity, who first proposed (in 1906) that the isomers of glyce-raldehyde be the standards for denoting the stereochemistry ofsugars and other molecules. Later, when experiments showed thatthe configuration of (�)-glyceraldehyde was related to (�)-glu-cose, (�)-glyceraldehyde was given the designation D. EmilFischer rejected the Rosanoff convention, but it was universallyaccepted. Ironically, this nomenclature system is often mistakenlyreferred to as the Fischer convention.The absolute configuration of (�)-glucose.H C OHCHOHO C HH C OHH C OHCH2OHFIGURE 4.13 ● The configuration of the common L-amino acids can be related to theconfiguration of L(�)-glyceraldehyde as shown. These drawings are known as Fischer pro-jections. The horizontal lines of the Fischer projections are meant to indicate bonds com-ing out of the page from the central carbon, and vertical lines represent bonds extendingbehind the page from the central carbon atom.C HHOCHOCH2OHL-GlyceraldehydeC OHHCHOCH2OHD-GlyceraldehydeC HH3NCOOHCH2OHL-SerineC NH3HCOOHCH2OHD-Serine+ +isomers of isoleucine are shown in Figure 4.14. The isomer obtained from digestsof natural proteins is arbitrarily designated L-isoleucine. In the (R,S) system, L-isoleucine is (2S,3S)-isoleucine. Its diastereomer is referred to as L-allo-isoleucine. The D-enantiomeric pair of isomers is named in a similar manner.98A D E E P E R L O O KThe Murchison Meteorite—Discovery of Extraterrestrial HandednessThe predominance of L-amino acids in biological systems is oneof life’s most intriguing features. Prebiotic syntheses of aminoacids would be expected to produce equal amounts of L- and D-enantiomers. Some kind of enantiomeric selection process musthave intervened to select L-amino acids over their D-counterpartsas the constituents of proteins. Was it random chance that choseL- over D-isomers?Analysis of carbon compounds—even amino acids—fromextraterrestrial sources might provide deeper insights into thismystery. John Cronin and Sandra Pizzarello have examined theenantiomeric distribution of unusual amino acids obtained fromthe Murchison meteorite, which struck the earth on September28, 1969, near Murchison, Australia. (By selecting unusual aminoacids for their studies, Cronin and Pizzarello ensured that theywere examining materials that were native to the meteorite andnot earth-derived contaminants.) Four �-dialkyl amino acids—�-methylisoleucine, �-methylalloisoleucine, �-methylnorvaline,and isovaline—were found to have an L-enantiomeric excess of 2to 9%.This may be the first demonstration that a natural L-enan-tiomer enrichment occurs in certain cosmological environments.Could these observations be relevant to the emergence of L-enan-tiomers as the dominant amino acids on the earth? And, if so,could there be life elsewhere in the universe that is based uponthe same amino acid handedness?*The four stereoisomers of this amino acid include the D- and L-forms of �-methylisoleucine and �-methylalloisoleucine.Cronin, J.R., and Pizzarello, S., 1997. Enantiomeric excesses in meteoritic amino acids. Science 275:951–955.C COOHCH2NH3�CH3CH3IsovalineC COOHCH2NH3�CH3CH3CH2�-MethylnorvalineCH C COOHCH2NH3�CH3CH3 CH32-Amino-2, 3-dimethylpentanoic acid*Amino acids found in the Murchison meteoriteFIGURE 4.14 ● The stereoisomers ofisoleucine and threonine. The structures at thefar left are the naturally occurring isomers.H3N HCOOHH3C HC2H5L-Isoleucine(2S,3S)-IsoleucineHCOOHHC2H5D-Isoleucine(2R,3R)-IsoleucineNH3CH3H3N HCOOHC2H5L-Alloisoleucine(2S,3R)-IsoleucineHCOOHC2H5D-Alloisoleucine(2R,3S)-IsoleucineNH3H CH3 H3C H+ + + +CCCCCCCCH3N HCOOHH OHCH3L-ThreonineHCOOHHOCH3D-ThreonineNH3HH3N HCOOHCH3L-AllothreonineHCOOHCH3D-AllothreonineNH3HO H H OH+ + + +CCCCCCCC4.5 ● Spectroscopic Properties of Amino AcidsOne of the most important and exciting advances in modern biochemistry hasbeen the application of spectroscopic methods, which measure the absorptionand emission of energy of different frequencies by molecules and atoms.Spectroscopic studies of proteins, nucleic acids, and other biomolecules areproviding many new insights into the structure and dynamic processes in thesemolecules.Ultraviolet SpectraMany details of the structure and chemistry of the amino acids have been elu-cidated or at least confirmed by spectroscopic measurements. None of theamino acids absorbs light in the visible region of the electromagnetic spec-trum. Several of the amino acids, however, do absorb ultraviolet radiation, andall absorb in the infrared region. The absorption of energy by electrons as theyrise to higher energy states occurs in the ultraviolet/visible region of the energyspectrum. Only the aromatic amino acids phenylalanine, tyrosine, and trypto-phan exhibit significant ultraviolet absorption above 250 nm, as shown in Figure4.15. These strong absorptions can be used for spectroscopic determinationsof protein concentration. The aromatic amino acids also exhibit relatively weakfluorescence, and it has recently been shown that tryptophan can exhibit phos-4.5 ● Spectroscopic Properties of Amino Acids 99C R I T I C A L D E V E L O P M E N T S I N B I O C H E M I S T R YRules for Description of Chiral Centers in the (R,S) SystemNaming a chiral center in the (R,S ) system is accomplished byviewing the molecule from the chiral center to the atom with thelowest priority. If the other three atoms facing the viewer thendecrease in priority in a clockwise direction, the center is said tohave the (R) configuration (where R is from the Latin rectusmeaning “right”). If the three atoms in question decrease in pri-ority in a counterclockwise fashion, the chiralcenter is of the (S )configuration (where S is from the Latin sinistrus meaning “left”).If two of the atoms coordinated to a chiral center are identical,the atoms bound to these two are considered for priorities. Forsuch purposes, the priorities of certain functional groups foundin amino acids and related molecules are in the following order:SH OH NH2 COOH CHO CH2OH CH3From this, it is clear that D-glyceraldehyde is (R)-glyceraldehyde,and L-alanine is (S)-alanine (see figure). Interestingly, the �-car-bon configuration of all the L-amino acids except for cysteine is (S).Cysteine, by virtue of its thiol group, is in fact (R )-cysteine.HO C HCHOCH2OHL-GlyceraldehydeHCH2OHOHCOH(S)-GlyceraldehydeHOHCHOCH2OHD-GlyceraldehydeHHOH2C CHOOH(R)-GlyceraldehydeCH3N HCOOHCH3L-AlanineCH3–OOCNH3(S)-Alanine+C H+The assignment of (R) and (S ) notation for glyceraldehyde and L-alanine.phorescence—a relatively long-lived emission of light. These fluorescence andphosphorescence properties are especially useful in the study of protein struc-ture and dynamics (see Chapter 6).Nuclear Magnetic Resonance SpectraThe development in the 1950s of nuclear magnetic resonance (NMR), a spec-troscopic technique that involves the absorption of radio frequency energy bycertain nuclei in the presence of a magnetic field, played an important part inthe chemical characterization of amino acids and proteins. Several importantprinciples rapidly emerged from these studies. First, the chemical shift1 ofamino acid protons depends on their particular chemical environment andthus on the state of ionization of the amino acid. Second, the change in elec-tron density during a titration is transmitted throughout the carbon chain inthe aliphatic amino acids and the aliphatic portions of aromatic amino acids,as evidenced by changes in the chemical shifts of relevant protons. Finally, themagnitude of the coupling constants between protons on adjacent carbonsdepends in some cases on the ionization state of the amino acid. This appar-ently reflects differences in the preferred conformations in different ioniza-tion states. Proton NMR spectra of two amino acids are shown in Figure 4.16.Because they are highly sensitive to their environment, the chemical shifts ofindividual NMR signals can detect the pH-dependent ionizations of aminoacids. Figure 4.17 shows the 13C chemical shifts occurring in a titration of lysine.Note that the chemical shifts of the carboxyl C, C�, and C� carbons of lysineare sensitive to dissociation of the nearby �-COOH and �-NH3� protons (withpKa values of about 2 and 9, respectively), whereas the C and C� carbons aresensitive to dissociation of the �-NH3� group. Such measurements have beenvery useful for studies of the ionization behavior of amino acid residues in pro-100 Chapter 4 ● Amino Acids1The chemical shift for any NMR signal is the difference in resonant frequency between theobserved signal and a suitable reference signal. If two nuclei are magnetically coupled, the NMRsignals of these nuclei split, and the separation between such split signals, known as the couplingconstant, is likewise dependent on the structural relationship between the two nuclei.FIGURE 4.15 ● The ultraviolet absorption spectra of the aromatic amino acids at pH 6.(From Wetlaufer, D.B., 1962. Ultraviolet spectra of proteins and amino acids. Advances in Protein Chemistry 17:303–390.)320300280260240220200Wavelength (nm)1020501002005001,0002,0005,00010,00020,00040,000Molar absorptivity, ε PheTyrTrpteins. More sophisticated NMR measurements at very high magnetic fields arealso used to determine the three-dimensional structures of peptides and evensmall proteins.4.6 ● Separation and Analysis of Amino Acid MixturesChromatographic MethodsThe purification and analysis of individual amino acids from complex mixtureswas once a very difficult process. Today, however, the biochemist has a widevariety of methods available for the separation and analysis of amino acids, orfor that matter, any of the other biological molecules and macromolecules we4.6 ● Separation and Analysis of Amino Acid Mixtures 101FIGURE 4.16 ● Proton NMR spectra of several amino acids. Zero on the chemical shiftscale is defined by the resonance of tetramethylsilane (TMS). (Adapted from Aldrich Library of NMR Spectra.)FIGURE 4.17 ● A plot of chemical shifts versus pH for the carbons of lysine. Changes inchemical shift are most pronounced for atoms near the titrating groups. Note the corre-spondence between the pKa values and the particular chemical shift changes. All chemicalshifts are defined relative to tetramethylsilane (TMS). (From Suprenant, H., et al., 1980. Journal of Magnetic Resonance 40:231–243.)Relative intensity910 8 7 6 5 4 3 2 1 0L-AlanineRelative intensity910 8 7 6 5 4 3 2 1 0L-Tyrosineppm ppmCH3H3NCOOHC H H3NCOOHC H CH2OH++6824101214pH4700 4500 4300 1400 1200 1000 800 600Chemical shift in Hz (vs. TMS)pK3pK2pK1carboxyl Cα ε β δ γencounter. All of these methods take advantage of the relative differences inthe physical and chemical characteristics of amino acids, particularly ioniza-tion behavior and solubility characteristics. The methods important for aminoacids include separations based on partition properties (the tendency to asso-ciate with one solvent or phase over another) and separations based on elec-trical charge. In all of the partition methods discussed here, the molecules ofinterest are allowed (or forced) to flow through a medium consisting of twophases—solid–liquid, liquid–liquid, or gas–liquid. In all of these methods, themolecules must show a preference for associating with one or the other phase.In this manner, the molecules partition, or distribute themselves, between thetwo phases in a manner based on their particular properties. The ratio of theconcentrations of the amino acid (or other species) in the two phases is des-ignated the partition coefficient.In 1903, a separation technique based on repeated partitioning betweenphases was developed by Mikhail Tswett for the separation of plant pigments(carotenes and chlorophylls). Tswett, a Russian botanist, poured solutions ofthe pigments through columns of finely divided alumina and other solid media,allowing the pigments to partition between the liquid solvent and the solid sup-port. Owing to the colorful nature of the pigments thus separated, Tswett calledhis technique chromatography. This term is now applied to a wide variety ofseparation methods, regardless of whether the products are colored or not.The success of all chromatography techniques depends on the repeated micro-scopic partitioning of a solute mixture between the available phases. The more frequently this partitioning can be made to occur within a given timespan or over a given volume, the more efficient is the resulting separation.Chromatographic methods have advanced rapidly in recent years, due in partto the development of sophisticated new solid-phase materials. Methods im-portant for amino acid separations include ion exchange chromatography, gas chromatography (GC), and high-performance liquid chromatography(HPLC).Ion Exchange ChromatographyThe separation of amino acids and other solutes is often achieved by means ofion exchange chromatography, in which the molecule of interest is exchangedfor another ion onto and off of a charged solid support. In a typical proce-dure, solutes in a liquid phase,usually water, are passed through columns filledwith a porous solid phase, usually a bed of synthetic resin particles, containingcharged groups. Resins containing positive charges attract negatively chargedsolutes and are referred to as anion exchangers. Solid supports possessing neg-ative charges attract positively charged species and are referred to as cationexchangers. Several typical cation and anion exchange resins with different typesof charged groups are shown in Figure 4.18. The strength of the acidity orbasicity of these groups and their number per unit volume of resin determinethe type and strength of binding of an exchanger. Fully ionized acidic groupssuch as sulfonic acids result in an exchanger with a negative charge which bindscations very strongly. Weakly acidic or basic groups yield resins whose charge(and binding capacity) depends on the pH of the eluting solvent. The choiceof the appropriate resin depends on the strength of binding desired. The barecharges on such solid phases must be counterbalanced by oppositely chargedions in solution (“counterions”). Washing a cation exchange resin, such asDowex-50, which has strongly acidic phenyl-SO3� groups, with a NaCl solutionresults in the formation of the so-called sodium form of the resin (see Figure4.19). When the mixture whose separation is desired is added to the column,the positively charged solute molecules displace the Na� ions and bind to the102 Chapter 4 ● Amino Acids4.6 ● Separation and Analysis of Amino Acid Mixtures 103FIGURE 4.18 ● Cation (a) and anion (b) exchange resins commonly used for bio-chemical separations.FIGURE 4.19 ● Operation of a cation exchange column, separating a mixture of Asp,Ser, and Lys. (a) The cation exchange resin in the beginning, Na� form. (b) A mixture ofAsp, Ser, and Lys is added to the column containing the resin. (c) A gradient of the elut-ing salt (e.g., NaCl) is added to the column. Asp, the least positively charged amino acid,is eluted first. (d) As the salt concentration increases, Ser is eluted. (e) As the salt concen-tration is increased further, Lys, the most positively charged of the three amino acids, iseluted last.StructureStrongly acidic, polystyrene resin (Dowex–50) S O–OOO CH2 CO–OWeakly acidic, carboxymethyl (CM) celluloseCH2 NCH2CCH2CWeakly acidic, chelating, polystyrene resin(Chelex–100)StructureStrongly basic, polystyrene resin (Dowex–1) CH3CH2 NCH3CH3Weakly basic, diethylaminoethyl (DEAE) celluloseHOCH2CH2 NCH2CH3CH2CH3+OOO–O–+(a) Cation Exchange Media(b) Anion Exchange MediaCation exchange beadbefore adding sampleBeadNa+ —SO3–Add mixture of Asp, Ser, LysAspLysSerAdd Na+ (NaCl)(c) Increase [Na+](d) Serine is eluted nextIncrease [Na+](e) (a) (b) Asp, the least positively charged amino acid, is eluted firstLysine, the most positively charged amino acid, is eluted lastresin. A gradient of an appropriate salt is then applied to the column, and thesolute molecules are competitively (and sequentially) displaced (eluted) fromthe column by the rising concentration of cations in the gradient, in an orderthat is inversely related to their affinities for the column. The separation of amixture of amino acids on such a column is shown in Figures 4.19 and 4.20.Figure 4.21, taken from a now-classic 1958 paper by Stanford Moore, DarrelSpackman, and William Stein, shows a typical separation of the common aminoacids. The events occurring in this separation are essentially those depicted inFigures 4.19 and 4.20. The amino acids are applied to the column at low pH(4.25), under which conditions the acidic amino acids (aspartate and gluta-mate, among others) are weakly bound and the basic amino acids, such as argi-nine and lysine, are tightly bound. Sodium citrate solutions, at two differentconcentrations and three different values of pH, are used to elute the aminoacids gradually from the column.A typical HPLC chromatogram using precolumn derivatization of aminoacids with o-phthaldialdehyde (OPA) is shown in Figure 4.22. HPLC has rapidlybecome the chromatographic technique of choice for most modern bio-chemists. The very high resolution, excellent sensitivity, and high speed of thistechnique usually outweigh the disadvantage of relatively low capacity.104 Chapter 4 ● Amino AcidsFIGURE 4.20 ● The separation of amino acids on a cation exchange column.Samplecontainingseveralamino acidsElutioncolumncontainingcation-exchangeresin beadsThe elution processseparates amino acidsinto discrete bandsEluant emergingfrom the columnis collectedAsp Ser LysAmino acidconcentrationElution timeSome fractionsdo not containamino acids4.6 ● Separation and Analysis of Amino Acid Mixtures 105FIGURE 4.21 ● Chromatographic fractionation of a synthetic mix-ture of amino acids on ion exchange columns using Amberlite IR-120, a sulfonated polystyrene resin similar to Dowex-50. A secondcolumn with different buffer conditions is used to resolve the basicamino acids. (Adapted from Moore, S., Spackman, D., and Stein, W., 1958.Chromatography of amino acids on sulfonated polystyrene resins. Analytical Chemistry30 :1185–1190.)FIGURE 4.22 ● HPLC chromatogram ofamino acids employing precolumn derivatiza-tion with OPA. Chromatography was carriedout on an Ultrasphere ODS column using acomplex tetrahydrofuran:methanol:0.05 Msodium acetate (pH 5.9) 1:19:80 tomethanol:0.05 M sodium acetate (pH 5.9) 4:1gradient at a flow rate of 1.7 mL/min.(Adapted from Jones, B. N., Pääbo, S., and Stein, S., 1981.Amino acid analysis and enzymic sequence determination of pep-tides by an improved o-phthaldialdehyde precolumn labeling pro-cedure. Journal of Liquid Chromatography 4:56–586.)0Volume of eluant 0.05Amount of solute0.100.150.200.250.3025 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475Phenyl-alanineTyrosineLeucineIsoleucineMethionineValineCystineAlanineGlycineProlineGlutamicacidSerineThreonineAsparticacidpH 3.250.2N Na citratepH 4.250.2N Na citrate0 25 50 75 100 125ArginineNH4+HistidineLysineTyrosinePhenylalaninepH 5.280.35N Na citrate0.050.100.150.200.250.30Amount of soluteVolume of eluant Relative fluorescence0Time (minutes)5 10 15 20 25 30 35100500% solvent BAsp GluAsn SerGln GlyThrArgβ-AlaAlaTyrTrpMetValPheIleLysPROBLEMS1. Without consulting chapter figures, draw Fischer projectionformulas for glycine, aspartate, leucine, isoleucine, methionine,and threonine.2. Without reference to the text, give the one-letter and three-letter abbreviations for asparagine, arginine, cysteine, lysine, pro-line, tyrosine, and tryptophan.3. Write equations for the ionic dissociations of alanine, gluta-mate, histidine, lysine, and phenylalanine.4. How is the pK a of the �-NH3� group affected by the presenceon an amino acid of the �-COO�?5. Draw an appropriate titration curve for aspartic acid, labelingthe axes and indicating the equivalence points and the pKa val-ues.6. Calculate the concentrations of all ionic species in a 0.25 Msolution of histidine at pH 2, pH 6.4, and pH 9.3.7. Calculate the pH at which the �-carboxyl group of glutamicacid is two-thirds dissociated.8. Calculate the pH at which the �-amino group of lysine is 20%dissociated.9. Calculate the pH of a 0.3 M solutionof (a) leucine hydrochlo-ride, (b) sodium leucinate, and (c) isoelectric leucine.10. Quantitative measurements of optical activity are usuallyexpressed in terms of the specific rotation, [�]D25, defined as[�]D25 �For any measurement of optical rotation, the wavelength of thelight used and the temperature must both be specified. In thiscase, D refers to the “D line” of sodium at 589 nm and 25 refers toa measurement temperature of 25°C. Calculate the concentrationof a solution of L-arginine that rotates the incident light by 0.35°in an optical path length of 1 dm (decimeter).11. Absolute configurations of the amino acids are referenced toD- and L-glyceraldehyde on the basis of chemical transformationsthat can convert the molecule of interest to either of these refer-ence isomeric structures. In such reactions, the stereochemicalconsequences for the asymmetric centers must be understood foreach reaction step. Propose a sequence of reactions that woulddemonstrate that L(�)-serine is stereochemically related to L(�)-glyceraldehyde.12. Describe the stereochemical aspects of the structure of cys-tine, the structure that is a disulfide-linked pair of cysteines.13. Draw a simple mechanism for the reaction of a cysteinesulfhydryl group with iodoacetamide.14. Describe the expected elution pattern for a mixture of aspar-tate, histidine, isoleucine, valine, and arginine on a column ofDowex-50.15. Assign (R,S ) nomenclature to the threonine isomers of Figure4.14.Measured rotation in degrees � 100(Optical path in dm) � (conc. in g�mL)106 Chapter 4 ● Amino AcidsFURTHER READINGBarker, R., 1971. Organic Chemistry of Biological Compounds, Chap. 4.Englewood Cliffs, NJ: Prentice-Hall.Barrett, G. C., ed., 1985. Chemistry and Biochemistry of the Amino Acids. NewYork: Chapman and Hall.Bovey, F. A., and Tiers, G. V. D., 1959. Proton N.S.R. spectroscopy. V. Studiesof amino acids and peptides in trifluoroacetic acid. Journal of the AmericanChemical Society 81:2870–2878.Cahn, R. S., 1964. An introduction to the sequence rule. Journal of ChemicalEducation 41:116–125.Greenstein, J. P., and Winitz, M., 1961. Chemistry of the Amino Acids. NewYork: John Wiley & Sons.Heiser, T., 1990. Amino acid chromatography: The “best” technique forstudent labs. Journal of Chemical Education 67:964–966.Herod, D. W., and Menzel, E. R., 1982. Laser detection of latent finger-prints: Ninhydrin. Journal of Forensic Science 27:200–204.Iizuka, E., and Yang, J. T., 1964. Optical rotatory dispersion of L-aminoacids in acid solution. Biochemistry 3:1519–1524.Kauffman, G. B., and Priebe, P. M., 1990. The Emil Fischer–WilliamRamsey friendship. Journal of Chemical Education 67:93–101.Mabbott, G., 1990. Qualitative amino acid analysis of small peptides byGC/MS. Journal of Chemical Education 67:441–445.Meister, A., 1965. Biochemistry of the Amino Acids, 2nd ed., Vol. 1. New York:Academic Press.Moore, S., Spackman, D., and Stein, W. H., 1958. Chromatography ofamino acids on sulfonated polystyrene resins. Analytical Chemistry 30:1185–1190.Roberts, G. C. K., and Jardetzky, O., 1970. Nuclear magnetic resonancespectroscopy of amino acids, peptides and proteins. Advances in ProteinChemistry 24:447–545.Segel, I. H., 1976. Biochemical Calculations, 2nd ed. New York: John Wiley& Sons.Suprenant, H. L., Sarneski, J. E., Key, R. R., Byrd, J. T., and Reilley, C. N., 1980. Carbon-13 NMR studies of amino acids: Chemical shifts, protona-tion shifts, microscopic protonation behavior. Journal of Magnetic Resonance40:231–243.Chapter 5Proteins: Their BiologicalFunctions and PrimaryStructure. . . by small and simple things are greatthings brought to pass.ALMA 37.6, The Book of Mormon107Proteins are a diverse and abundant class of biomolecules, constituting morethan 50% of the dry weight of cells. This diversity and abundance reflect thecentral role of proteins in virtually all aspects of cell structure and function.An extraordinary diversity of cellular activity is possible only because of the ver-satility inherent in proteins, each of which is specifically tailored to its biolog-ical role. The pattern by which each is tailored resides within the genetic infor-mation of cells, encoded in a specific sequence of nucleotide bases in DNA.Although helices are uncommon in manmade architecture,they are a common structural theme in biological macromole-cules—proteins, nucleic acids, and even polysaccharides.(Loretto Chapel, Santa Fe, NM/ © Sarbo)OUTLINE5.1 ● Proteins Are Linear Polymers of Amino Acids5.2 ● Architecture of Protein Molecules5.3 ● The Many Biological Functions of Proteins5.4 ● Some Proteins Have Chemical GroupsOther Than Amino Acids5.5 ● Reactions of Peptides and Proteins5.6 ● Purification of Protein Mixtures5.7 ● The Primary Structure of a Protein:Determining the Amino Acid Sequence5.8 ● Nature of Amino Acid Sequences5.9 ● Synthesis of Polypeptides in the LaboratoryEach such segment of encoded information defines a gene, and expression ofthe gene leads to synthesis of the specific protein encoded by it, endowing thecell with the functions unique to that particular protein. Proteins are the agentsof biological function; they are also the expressions of genetic information.5.1 ● Proteins Are Linear Polymers of Amino AcidsChemically, proteins are unbranched polymers of amino acids linked head totail, from carboxyl group to amino group, through formation of covalent pep-tide bonds, a type of amide linkage (Figure 5.1).Peptide bond formation results in the release of H2O. The peptide “back-bone” of a protein consists of the repeated sequence ONOC�OCO, wherethe N represents the amide nitrogen, the C� is the �-carbon atom of an aminoacid in the polymer chain, and the final C is the carbonyl carbon of the aminoacid, which in turn is linked to the amide N of the next amino acid down theline. The geometry of the peptide backbone is shown in Figure 5.2. Note thatthe carbonyl oxygen and the amide hydrogen are trans to each other in thisfigure. This conformation is favored energetically because it results in less sterichindrance between nonbonded atoms in neighboring amino acids. Becausethe �-carbon atom of the amino acid is a chiral center (in all amino acidsexcept glycine), the polypeptide chain is inherently asymmetric. Only L-aminoacids are found in proteins.The Peptide Bond Has Partial Double Bond CharacterThe peptide linkage is usually portrayed by a single bond between the carbonylcarbon and the amide nitrogen (Figure 5.3a). Therefore, in principle, rotationmay occur about any covalent bond in the polypeptide backbone because allthree kinds of bonds (NOC�, C�OCo, and the CoON peptide bond) are sin-gle bonds. In this representation, the C and N atoms of the peptide groupingare both in planar sp2 hybridization and the C and O atoms are linked by a �bond, leaving the nitrogen with a lone pair of electrons in a 2p orbital. However,another resonance form for the peptide bond is feasible in which the C andN atoms participate in a � bond, leaving a lone e� pair on the oxygen (Figure5.3b). This structure prevents free rotation about the CoON peptide bondbecause it becomes a double bond. The real nature of the peptide bond liessomewhere between these extremes; that is, it has partial double bond char-acter, as represented by the intermediate form shown in Figure 5.3c.Peptide bond resonance has several important consequences. First, itrestricts free rotation around the peptide bond and leaves the peptide back-bone with only two degrees of freedom per amino acid group: rotation around108 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureFIGURE 5.1 ● Peptide formation is the creation of anamide bond between the carboxylgroup of one amino acid and the amino group of another amino acid. R1 and R2 repre-sent the R groups of two different amino acids.CCHOO–H3NONR1CAmino acid 1+ CHOO–H3NR2CAmino acid 2CHH3NR1DipeptideOO–CHCHR2H2O+ + +the NOC� bond and rotation around the C�OCo bond.1 Second, the six atomscomposing the peptide bond group tend to be coplanar, forming the so-calledamide plane of the polypeptide backbone (Figure 5.4). Third, the CoON bondlength is 0.133 nm, which is shorter than normal CON bond lengths (for exam-ple, the C�ON bond of 0.145 nm) but longer than typical CPN bonds (0.125nm). The peptide bond is estimated to have 40% double-bond character.FIGURE 5.2 ● The peptide bond is shown inits usual trans conformation of carbonyl O andamide H. The C� atoms are the �-carbons oftwo adjacent amino acids joined in peptidelinkage. The dimensions and angles are theaverage values observed by crystallographicanalysis of amino acids and small peptides. Thepeptide bond is the light gray bond between Cand N. (Adapted from Ramachandran, G. N., et al., 1974.Biochimica Biophysica Acta 359:298–302.)5.1 ● Proteins Are Linear Polymers of Amino Acids 109FIGURE 5.3 ● The partial double bond char-acter of the peptide bond. Resonance interac-tions among the carbon, oxygen, and nitrogenatoms of the peptide group can be representedby two resonance extremes (a and b). (a) Theusual way the peptide atoms are drawn. (b) Inan equally feasible form, the peptide bond isnow a double bond; the amide N bears a posi-tive charge and the carbonyl O has a negativecharge. (c) The actual peptide bond is bestdescribed as a resonance hybrid of the forms in(a) and (b). Significantly, all of the atoms asso-ciated with the peptide group are coplanar,rotation about CoON is restricted, and the peptide is distinctly polar. (Irving Geis)RCαRC0.123nm115.6�123.2�0.133nm121.1�HH0.1nm121.9�119.5�N0.145nm118.2�Cα0.152nmOH(a)NCHCαCαOC NCαCαHOA pure double bond between Cand O would permit free rotationaround the C N bond.NCHCαCαO(b)C NCαCαH–O+The other extreme would prohibitC N bond rotation but wouldplace too great a charge onO and N.NCHCαCαOThe true electron density isintermediate. The barrier toC N bond rotation of about 88 kJ/mol is enough tokeep the amide group planar.(c)1The angle of rotation about the NOC� bond is designated �, phi, whereas the C�OCo angle ofrotation is designated �, psi.The Polypeptide Backbone Is Relatively PolarPeptide bond resonance also causes the peptide backbone to be relatively polar.As shown in Figure 5.3b, the amide nitrogen represents a protonated or posi-tively charged form, and the carbonyl oxygen becomes a negatively chargedatom in the double-bonded resonance state. In actuality, the hybrid state of thepartially double-bonded peptide arrangement gives a net positive charge of0.28 on the amide N and an equivalent net negative charge of 0.28 on the car-bonyl O. The presence of these partial charges means that the peptide bondhas a permanent dipole. Nevertheless, the peptide backbone is relatively unre-active chemically, and protons are gained or lost by the peptide groups only atextreme pH conditions.Peptide ClassificationPeptide is the name assigned to short polymers of amino acids. Peptides areclassified by the number of amino acid units in the chain. Each unit is calledan amino acid residue, the word residue denoting what is left after the releaseof H2O when an amino acid forms a peptide link upon joining the peptidechain. Dipeptides have two amino acid residues, tripeptides have three,tetrapeptides four, and so on. After about 12 residues, this terminology becomescumbersome, so peptide chains of more than 12 and less than about 20 aminoacid residues are usually referred to as oligopeptides, and, when the chainexceeds several dozen amino acids in length, the term polypeptide is used. Thedistinctions in this terminology are not precise.Proteins Are Composed of One or More Polypeptide ChainsThe terms polypeptide and protein are used interchangeably in discussing singlepolypeptide chains. The term protein broadly defines molecules composed ofone or more polypeptide chains. Proteins having only one polypeptide chainare monomeric proteins. Proteins composed of more than one polypeptidechain are multimeric proteins. Multimeric proteins may contain only one kindof polypeptide, in which case they are homomultimeric, or they may be com-posed of several different kinds of polypeptide chains, in which instance theyare heteromultimeric. Greek letters and subscripts are used to denote thepolypeptide composition of multimeric proteins. Thus, an �2-type protein is adimer of identical polypeptide subunits, or a homodimer. Hemoglobin (Table5.1) consists of four polypeptides of two different kinds; it is an �2�2 hetero-multimer.FIGURE 5.4 ● The coplanar relationship ofthe atoms in the amide group is highlighted asan imaginary shaded plane lying between twosuccessive �-carbon atoms in the peptide back-bone.110 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureOα-carbonRCCHNα-carbonHCRH5.1 ● Proteins Are Linear Polymers of Amino Acids 111Table 5.1Size of Protein Molecules*Protein Mr Number of Residues per Chain Subunit OrganizationInsulin (bovine) 5,733 21 (A) ��30 (B)Cytochrome c (equine) 12,500 104 �1Ribonuclease A (bovine pancreas) 12,640 124 �1Lysozyme (egg white) 13,930 129 �1Myoglobin (horse) 16,980 153 �1Chymotrypsin (bovine pancreas) 22,600 13 (�) ���132 (�)97 (�)Hemoglobin (human) 64,500 141 (�) �2�2146 (�)Serum albumin (human) 68,500 550 �1Hexokinase (yeast) 96,000 200 �4�-Globulin (horse) 149,900 214 (�) �2�2446 (�)Glutamate dehydrogenase (liver) 332,694 500 �6Myosin (rabbit) 470,000 1800 (heavy, h) h2�1��2�2190 (�)149 (��)160 (�)Ribulose bisphosphate carboxylase (spinach) 560,000 475 (�) �8�8123 (�)Glutamine synthetase (E. coli) 600,000 468 �12*Illustrations of selected proteins listed in Table 5.1 are drawn to constant scale. Adapted from Goodsell and Olson, 1993. Trends in Biochemical Sciences 18:65–68.InsulinCytochrome c RibonucleaseLysozyme MyoglobinGlutamine synthetaseImmunoglobulinHemoglobinPolypeptide chains of proteins range in length from about 100 amino acidsto 1800, the number found in each of the two polypeptide chains of myosin,the contractile protein of muscle. However, titin, another muscle protein, hasnearly 27,000 amino acid residues and a molecular weight of 2.8 � 106. Theaverage molecular weight of polypeptide chains in eukaryotic cells is about31,700, corresponding to about 270 amino acid residues. Table 5.1 is a repre-sentative list of proteins according to size. The molecular weights (Mr) of pro-teins can be estimated by a number of physicochemical methods such as poly-acrylamide gel electrophoresis or ultracentrifugation (see Chapter Appendix).Precise determinations of protein molecular masses are best obtained by simplecalculations based on knowledge of their amino acid sequence. No simple gen-eralizations correlate the size of proteins with their functions. For instance, thesame function may be fulfilled in different cells by proteins of different mole-cular weight. The Escherichia coli enzyme responsible for glutamine synthesis (aprotein known as glutamine synthetase) has a molecular weight of 600,000,whereas the analogousenzyme in brain tissue has a molecular weight of just380,000.Acid Hydrolysis of ProteinsPeptide bonds of proteins are hydrolyzed by either strong acid or strong base.Because acid hydrolysis proceeds without racemization and with less destruc-tion of certain amino acids (Ser, Thr, Arg, and Cys) than alkaline treatment,it is the method of choice in analysis of the amino acid composition of pro-teins and polypeptides. Typically, samples of a protein are hydrolyzed with 6 NHCl at 110°C for 24, 48, and 72 hr in sealed glass vials. Tryptophan is destroyedby acid and must be estimated by other means to determine its contributionto the total amino acid composition. The OH-containing amino acids serineand threonine are slowly destroyed, but the data obtained for the three timepoints (24, 48, and 72 hr) allow extrapolation to zero time to estimate the orig-inal Ser and Thr content (Figure 5.5). In contrast, peptide bonds involvinghydrophobic residues such as valine and isoleucine are only slowly hydrolyzedin acid. Another complication arises because the �- and �-amide linkages inasparagine (Asn) and glutamine (Gln) are acid labile. The amino nitrogen isreleased as free ammonium, and all of the Asn and Gln residues of the pro-tein become aspartic acid (Asp) and glutamic acid (Glu), respectively. The112 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureFIGURE 5.5 ● (a) The hydroxy amino acids serine and threonine are slowly destroyedduring the course of protein hydrolysis for amino acid composition analysis. Extrapolationof the data back to time zero allows an accurate estimation of the amount of these aminoacids originally present in the protein sample. (b) Peptide bonds involving hydrophobicamino acid residues such as valine and isoleucine resist hydrolysis by HCl. With time,these amino acids are released and their free concentrations approach a limiting valuethat can be approximated with reliability.(a) (b)Time100110Time[Free amino acids] as %present in protein100050Serine, threonine% original amino acid remainingHydrophobic amino acids,e.g., valine, isoleucineamount of ammonium released during acid hydrolysis gives an estimate of thetotal number of Asn and Gln residues in the original protein, but not theamounts of either. Accordingly, the concentrations of Asp and Glu determinedin amino acid analysis are expressed as Asx and Glx, respectively. Because therelative contributions of [Asn Asp] or [Gln Glu] cannot be derived fromthe data, this information must be obtained by alternative means.Amino Acid Analysis of ProteinsThe complex amino acid mixture in the hydrolysate obtained after digestionof a protein in 6 N HCl can be separated into the component amino acids byeither ion exchange chromatography (see Chapter 4) or by reversed-phasehigh-pressure liquid chromatography (HPLC) (see Chapter Appendix). Theamount of each amino acid can then be determined. In ion exchange chro-matography, the amino acids are separated and then quantified following reac-tion with ninhydrin (so-called postcolumn derivatization). In HPLC, the aminoacids are converted to phenylthiohydantoin (PTH) derivatives via reaction withEdman’s reagent (see Figure 5.19) prior to chromatography (precolumnderivatization). Both of these methods of separation and analysis are fully auto-mated in instruments called amino acid analyzers. Analysis of the amino acidcomposition of a 30-kD protein by these methods requires less than 1 hour andonly 6 g (0.2 nmol) of the protein.Table 5.2 gives the amino acid composition of several selected proteins:ribonuclease A, alcohol dehydrogenase, myoglobin, histone H3, and collagen.Each of the 20 naturally occurring amino acids is usually represented at leastonce in a polypeptide chain. However, some small proteins may not have a rep-resentative of every amino acid. Note that ribonuclease (12.6 kD, 124 aminoacid residues) does not contain any tryptophan. Amino acids almost neveroccur in equimolar ratios in proteins, indicating that proteins are not com-posed of repeating arrays of amino acids. There are a few exceptions to thisrule. Collagen, for example, contains large proportions of glycine and proline,and much of its structure is composed of (Gly-x -Pro) repeating units, where xis any amino acid. Other proteins show unusual abundances of various aminoacids. For example, histones are rich in positively charged amino acids such asarginine and lysine. Histones are a class of proteins found associated with theanionic phosphate groups of eukaryotic DNA.Amino acid analysis itself does not directly give the number of residues ofeach amino acid in a polypeptide, but it does give amounts from which thepercentages or ratios of the various amino acids can be obtained (Table 5.2).If the molecular weight and the exact amount of the protein analyzed are known(or the number of amino acid residues per molecule is known), the molarratios of amino acids in the protein can be calculated. Amino acid analysis pro-vides no information on the order or sequence of amino acid residues in thepolypeptide chain. Because the polypeptide chain is unbranched, it has onlytwo ends, an amino-terminal or N-terminal end and a carboxyl-terminal or C-terminal end.The Sequence of Amino Acids in ProteinsThe unique characteristic of each protein is the distinctive sequence of aminoacid residues in its polypeptide chain(s). Indeed, it is the amino acid sequenceof proteins that is encoded by the nucleotide sequence of DNA. This aminoacid sequence, then, is a form of genetic information. By convention, the aminoacid sequence is read from the N-terminal end of the polypeptide chainthrough to the C-terminal end. As an example, every molecule of ribonucle-5.1 ● Proteins Are Linear Polymers of Amino Acids 113ase A from bovine pancreas has the same amino acid sequence, beginning withN-terminal lysine at position 1 and ending with C-terminal valine at position124 (Figure 5.6). Given the possibility of any of the 20 amino acids at eachposition, the number of unique amino acid sequences is astronomically large.The astounding sequence variation possible within polypeptide chains provides114 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureTable 5.2Amino Acid Composition of Some Selected ProteinsValues expressed are percent representation of each amino acid.Proteins*Amino Acid RNase ADH Mb Histone H3 CollagenAla 6.9 7.5 9.8 13.3 11.7Arg 3.7 3.2 1.7 13.3 4.9Asn 7.6 2.1 2.0 0.7 1.0Asp 4.1 4.5 5.0 3.0 3.0Cys 6.7 3.7 0 1.5 0Gln 6.5 2.1 3.5 5.9 2.6Glu 4.2 5.6 8.7 5.2 4.5Gly 3.7 10.2 9.0 5.2 32.7His 3.7 1.9 7.0 1.5 0.3Ile 3.1 6.4 5.1 5.2 0.8Leu 1.7 6.7 11.6 8.9 2.1Lys 7.7 8.0 13.0 9.6 3.6Met 3.7 2.4 1.5 1.5 0.7Phe 2.4 4.8 4.6 3.0 1.2Pro 4.5 5.3 2.5 4.4 22.5Ser 12.2 7.0 3.9 3.7 3.8Thr 6.7 6.4 3.5 7.4 1.5Trp 0 0.5 1.3 0 0Tyr 4.0 1.1 1.3 2.2 0.5Val 7.1 10.4 4.8 4.4 1.7Acidic 8.4 10.2 13.7 8.1 7.5Basic 15.0 13.1 21.8 24.4 8.8Aromatic 6.4 6.4 7.2 5.2 1.7Hydrophobic 18.0 30.7 27.6 23.0 6.5*Proteins are as follows:RNase: Bovine ribonuclease A, an enzyme; 124 amino acid residues. Note that RNase lackstryptophan.ADH: Horse liver alcohol dehydrogenase, an enzyme; dimer of identical 374 amino acidpolypeptide chains. The amino acid composition of ADH is reasonably representative of thenorm for water-soluble proteins.Mb: Sperm whale myoglobin, an oxygen-binding protein; 153 amino acid residues. Note thatMb lacks cysteine.Histone H3: Histones are DNA-binding proteins found in chromosomes; 135 amino acidresidues. Note the very basic nature of this protein due to its abundance ofArg and Lysresidues. It also lacks tryptophan.Collagen: Collagen is an extracellular structural protein; 1052 amino acid residues. Collagenhas an unusual amino acid composition; it is about one-third glycine and is rich in proline.Note that it also lacks Cys and Trp and is deficient in aromatic amino acid residues in general.a key insight into the incredible functional diversity of protein molecules inbiological systems, which is discussed shortly.5.2 ● Architecture of Protein MoleculesProtein ShapeAs a first approximation, proteins can be assigned to one of three global classeson the basis of shape and solubility: fibrous, globular, or membrane (Figure5.7). Fibrous proteins tend to have relatively simple, regular linear structures.These proteins often serve structural roles in cells. Typically, they are insolu-ble in water or in dilute salt solutions. In contrast, globular proteins are roughlyspherical in shape. The polypeptide chain is compactly folded so that hydropho-bic amino acid side chains are in the interior of the molecule and thehydrophilic side chains are on the outside exposed to the solvent, water.Consequently, globular proteins are usually very soluble in aqueous solutions.Most soluble proteins of the cell, such as the cytosolic enzymes, are globularin shape. Membrane proteins are found in association with the various mem-brane systems of cells. For interaction with the nonpolar phase within mem-branes, membrane proteins have hydrophobic amino acid side chains orientedoutward. As such, membrane proteins are insoluble in aqueous solutions butcan be solubilized in solutions of detergents. Membrane proteins characteris-tically have fewer hydrophilic amino acids than cytosolic proteins.5.2 ● Architecture of Protein Molecules 115Val Ser AlaAspPheHisValProValTyrProAsnGlyGluAlaValIleIleHisLys Asn Ala Gln ThrLysThrTyrAlaCysAsnProTyrLysSerSerGlyThrGluArgCysAspThrIleSerMetThrSerTyrSerGlnTyrCysAsnThrGlnGlyAsnLysCysAlaValAsnLysGlnSerValAlaGlnValAspAlaLeu SerGlu His Val Phe Thr AsnValProLysCysArgAspLysThrLeuAsnArgSerLysMetMetGlnAsnCysTyrAsnSerSerSerAlaAlaSer Thr SerSer Asp Met His Gln ArgGluPheLysAlaAlaAlaThrGluLysH2N 17101272656058504140959030119120124HOOCCysCys110802021708426100FIGURE 5.6 ● Bovine pancreatic ribonuclease A contains 124 amino acid residues, noneof which are tryptophan. Four intrachain disulfide bridges (SOS) form cross-links in thispolypeptide between Cys26 and Cys84, Cys40 and Cys95, Cys58 and Cys110, and Cys65 andCys72. These disulfides are depicted by yellow bars.116 Chapter 5 ● Proteins: Their Biological Functions and Primary Structure(a)Collagen, afibrous protein(b) (c)Myoglobin, a globular proteinNH3+COO–BacteriorhodopsinPhospholipidmembraneFIGURE 5.7 ● (a) Proteins having structural roles in cells are typically fibrous and oftenwater insoluble. Collagen is a good example. Collagen is composed of three polypeptidechains that intertwine. (b) Soluble proteins serving metabolic functions can be character-ized as compactly folded globular molecules, such as myoglobin. The folding pattern putshydrophilic amino acid side chains on the outside and buries hydrophobic side chains inthe interior, making the protein highly water soluble. (c) Membrane proteins fold so thathydrophobic amino acid side chains are exposed in their membrane-associated regions.The portions of membrane proteins extending into or exposed at the aqueous environ-ments are hydrophilic in character, like soluble proteins. Bacteriorhodopsin is a typicalmembrane protein; it binds the light-absorbing pigment, cis-retinal, shown here in red. (a, b, Irving Geis)A D E E P E R L O O KThe Virtually Limitless Number of Different Amino Acid SequencesGiven 20 different amino acids, a polypeptide chain of n residuescan have any one of 20n possible sequence arrangements. To por-tray this, consider the number of tripeptides possible if there wereonly three different amino acids, A, B, and C (tripeptide � 3 � n;n3 � 33 � 27):AAA BBB CCCAAB BBA CCAAAC BBC CCBABA BAB CBCACA BCB CACABC BAA CBAACB BCC CABABB BAC CBBACC BCA CAAFor a polypeptide chain of 100 residues in length, a rather mod-est size, the number of possible sequences is 20100, or because 20 � 101.3, 10130 unique possibilities. These numbers are morethan astronomical! Because an average protein molecule of 100residues would have a mass of 13,800 daltons (average molecularmass of an amino acid residue � 138), 10130 such moleculeswould have a mass of 1.38 � 10134 daltons. The mass of the observ-able universe is estimated to be 1080 proton masses (about 1080daltons). Thus, the universe lacks enough material to make justone molecule of each possible polypeptide sequence for a pro-tein only 100 residues in length.The Levels of Protein StructureThe architecture of protein molecules is quite complex. Nevertheless, this com-plexity can be resolved by defining various levels of structural organization.Primary StructureThe amino acid sequence is the primary (1°) structure of a protein, such asthat shown in Figure 5.6, for example.Secondary StructureThrough hydrogen bonding interactions between adjacent amino acid residues(discussed in detail in Chapter 6), the polypeptide chain can arrange itself intocharacteristic helical or pleated segments. These segments constitute structuralconformities, so-called regular structures, that extend along one dimension,like the coils of a spring. Such architectural features of a protein are desig-nated secondary (2°) structures (Figure 5.8). Secondary structures are just oneof the higher levels of structure that represent the three-dimensional arrange-ment of the polypeptide in space.FIGURE 5.8 ● Two structural motifs thatarrange the primary structure of proteins intoa higher level of organization predominate inproteins: the �-helix and the �-pleated strand.Atomic representations of these secondarystructures are shown here, along with the sym-bols used by structural chemists to representthem: the flat, helical ribbon for the �-helixand the flat, wide arrow for �-structures. Bothof these structures owe their stability to the for-mation of hydrogen bonds between NOH andOPC functions along the polypeptide back-bone (see Chapter 6).5.2 ● Architecture of Protein Molecules 117 -HelixOnly the N C backboneis represented. The vertical line is the helix axis.CCNNCNCNCCNNNCCNC N CNNC -StrandThe N CO backbone as wellas the of R groups are represented here. Note that the amide planesare perpendicular to the page.NOCNCCCCαCαCαCαCαCαCαCαCαCαCαCβCβCβCβCβCβCβCαCαCαCαCαCαCαCαCOCNCHCNCNOCC“Shorthand” -helix “Shorthand” -strandHααββ118 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureTertiary StructureWhen the polypeptide chains of protein molecules bend and fold in order toassume a more compact three-dimensional shape, a tertiary (3°) level of struc-ture is generated (Figure 5.9). It is by virtue of their tertiary structure that pro-teins adopt a globular shape. A globular conformation gives the lowest surface-to-volume ratio,minimizing interaction of the protein with the surroundingenvironment.Quaternary StructureMany proteins consist of two or more interacting polypeptide chains of char-acteristic tertiary structure, each of which is commonly referred to as a sub-unit of the protein. Subunit organization constitutes another level in the hier-archy of protein structure, defined as the protein’s quaternary (4°) structure(Figure 5.10). Questions of quaternary structure address the various kinds ofsubunits within a protein molecule, the number of each, and the ways in whichthey interact with one another.Whereas the primary structure of a protein is determined by the covalentlylinked amino acid residues in the polypeptide backbone, secondary and higherH2N–CGVPAIQPVL10SGL[SR]IVNGE20EAVPGSWPWQ30VSLQDKTGFH40GGSLINEN50WVVTAAHCGV60TTSDVVVAGE70FDQGSSSEKI80QKLKIAKVFK90NSKYNSLTIN100NDITLLKLST110AASFSQTVSA120VCLPSASDDF130AAGTTCVTTG140WGLTRY[TN]AN150LPSDRLQQASL160PLLSNTNCKK170YWGTKIKDAM180ICAGASGVSS190CMGDSGGPLV200CKKNGAWTLV210GIVSWGSSTC220STSTPGVYAR230VTALVNWVQQ240TLAAN–COOH(a)—Chymotrypsin primary structure(b)—Chymotrypsin tertiary structureChymotrypsin space-filling model2191461491547519021352520115291CN2402341271781741701612233682641098710594981302276049184245Chymotrypsin ribbonFIGURE 5.9 ● Folding of the polypeptidechain into a compact, roughly spherical confor-mation creates the tertiary level of proteinstructure. (a) The primary structure and (b) a representation of the tertiary structure ofchymotrypsin, a proteolytic enzyme, are shownhere. The tertiary representation in (b) showsthe course of the chymotrypsin folding patternby successive numbering of the amino acids inits sequence. (Residues 14 and 15 and 147 and148 are missing because these residues areremoved when chymotrypsin is formed from itslarger precursor, chymotrypsinogen.) The rib-bon diagram depicts the three-dimensionaltrack of the polypeptide in space.orders of structure are determined principally by noncovalent forces such ashydrogen bonds and ionic, van der Waals, and hydrophobic interactions. It isimportant to emphasize that all the information necessary for a protein molecule toachieve its intricate architecture is contained within its 1° structure, that is, within theamino acid sequence of its polypeptide chain(s). Chapter 6 presents a detaileddiscussion of the 2°, 3°, and 4° structure of protein molecules.Protein ConformationThe overall three-dimensional architecture of a protein is generally referredto as its conformation. This term is not to be confused with configuration,which denotes the geometric possibilities for a particular set of atoms (Figure5.11). In going from one configuration to another, covalent bonds must bebroken and rearranged. In contrast, the conformational possibilities of a mole-cule are achieved without breaking any covalent bonds. In proteins, rotationsabout each of the single bonds along the peptide backbone have the potentialto alter the course of the polypeptide chain in three-dimensional space. Theserotational possibilities create many possible orientations for the protein chain,referred to as its conformational possibilities. Of the great number of theoreticalconformations a given protein might adopt, only a very few are favored ener-getically under physiological conditions. At this time, the rules that direct thefolding of protein chains into energetically favorable conformations are stillnot entirely clear; accordingly, they are the subject of intensive contemporaryresearch.FIGURE 5.10 ● Hemoglobin, which consistsof two � and two � polypeptide chains, is anexample of the quaternary level of proteinstructure. In this drawing, the �-chains are thetwo uppermost polypeptides and the two �-chains are the lower half of the molecule. Thetwo closest chains (darkest colored) are the �2-chain (upper left) and the �1-chain (lower right).The heme groups of the four globin chains arerepresented by rectangles with spheres (theheme iron atom). Note the symmetry of thismacromolecular arrangement. (Irving Geis)5.2 ● Architecture of Protein Molecules 119FIGURE 5.11 ● Configuration and conformation are not synonymous. (a) Rearrange-ments between configurational alternatives of a molecule can be achieved only by break-ing and remaking bonds, as in the transformation between the D- and L-configurationsof glyceraldehyde. No possible rotational reorientation of bonds linking the atoms of D-glyceraldehyde yields geometric identity with L-glyceraldehyde, even though they are mir-ror images of each other. (b) The intrinsic free rotation around single covalent bonds cre-ates a great variety of three-dimensional conformations, even for relatively simple mole-cules. Consider 1,2-dichloroethane. Viewed end-on in a Newman projection, threeprincipal rotational orientations or conformations predominate. Steric repulsion betweeneclipsed and partially eclipsed conformations keeps the possibilities at a reasonable num-ber. (c) Imagine the conformational possibilities for a protein in which two of every threebonds along its backbone are freely rotating single bonds.ClH H(a) CHOCH2OHOHHCHOCH2OHHO HC CD-Glyceraldehyde L-Glyceraldehyde(b)CCHClHHHCl1,2–DichloroethaneCH ClCHCClHHClHHClHHClHH(c)CNHCOCNHHAmino acidsSide chainAmide planesCOC-Chains�-Chains�HemeLater we return to an analysis of the 1° structure of proteins and themethodology used in determining the amino acid sequence of polypeptidechains, but let’s first consider the extraordinary variety and functional diver-sity of these most interesting macromolecules.5.3 ● The Many Biological Functions of ProteinsProteins are the agents of biological function. Virtually every cellular activity is depen-dent on one or more particular proteins. Thus, a convenient way to classify theenormous number of proteins is by the biological roles they fill. Table 5.3 sum-marizes the classification of proteins by function and gives examples of repre-sentative members of each class.EnzymesBy far the largest class of proteins is enzymes. More than 3000 different enzymesare listed in Enzyme Nomenclature, the standard reference volume on enzymeclassification. Enzymes are catalysts that accelerate the rates of biological reac-tions. Each enzyme is very specific in its function and acts only in a particularmetabolic reaction. Virtually every step in metabolism is catalyzed by an enzyme.The catalytic power of enzymes far exceeds that of synthetic catalysts. Enzymescan enhance reaction rates in cells as much as 1016 times the uncatalyzed rate.Enzymes are systematically classified according to the nature of the reactionthat they catalyze, such as the transfer of a phosphate group (phosphotransferase)or an oxidation–reduction (oxidoreductase). The formal names of enzymes come from the particular reaction within the class that they catalyze, as in ATP:D-fructose-6-phosphate 1-phosphotransferase and alcohol:NAD oxido-reductase. Often, enzymes have common names in addition to their formalnames. ATP:D-fructose-6-phosphate 1-phosphotransferase is more commonlyknown as phosphofructokinase (kinase is a common name given to ATP-depen-dent phosphotransferases). Similarly, alcohol:NAD oxidoreductase is casuallyreferred to as alcohol dehydrogenase. The reactions catalyzed by these two enzymesare shown in Figure 5.12. Other enzymes are known by trivial names that havehistorical roots, such as catalase (systematic name, hydrogen-peroxide:hydro-gen-peroxideoxidoreductase), and sometimes these trivial names have descrip-tive connotations as well, as in malic enzyme (systematic name, L-malate:NADPoxidoreductase).FIGURE 5.12 ● Enzymes are classifiedaccording to the specific biological reactionthat they catalyze. Cells contain thousands ofdifferent enzymes. Two common examplesdrawn from carbohydrate metabolism are phos-phofructokinase (PFK), or, more precisely, ATP : D-fructose-6-phosphate 1-phosphotrans-ferase, and alcohol dehydrogenase (ADH), oralcohol : NAD oxidoreductase, which catalyzethe reactions shown here.120 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureOHPhosphofructokinase (PFK)2–O3POH2C O CH2OHOHHH OH2–O3POH2C O CH2OPO32–OHHHHPFKATP + D - fructose-6-phosphate ADP + D - fructose-1,6-bisphosphateAlcohol dehydrogenase (ADH)NAD+ + CH3CH2OH NADH + H+ + CH3CHOEthyl alcoholAcetaldehydeADHH HOHO5.3 ● The Many Biological Functions of Proteins 121Table 5.3Biological Functions of Proteins and Some Representative ExamplesFunctional Class ExamplesEnzymes RibonucleaseTrypsinPhosphofructokinaseAlcohol dehydrogenaseCatalase“Malic” enzymeRegulatory proteins InsulinSomatotropinThyrotropinlac repressorNF1 (nuclear factor 1)Catabolite activator protein (CAP)AP1Transport proteins HemoglobinSerum albuminGlucose transporterStorage proteins OvalbuminCaseinZeinPhaseolinFerritinContractile and motile proteins ActinMyosinTubulinDyneinKinesinStructural proteins �-KeratinCollagenElastinFibroinProteoglycansScaffold proteins Grb 2crkshcstatIRS-1Protective and exploitive proteins ImmunoglobulinsThrombinFibrinogenAntifreeze proteinsSnake and bee venom proteinsDiphtheria toxinRicinExotic proteins MonellinResilinGlue proteinsRegulatory ProteinsA number of proteins do not perform any obvious chemical transformationbut nevertheless can regulate the ability of other proteins to carry out theirphysiological functions. Such proteins are referred to as regulatory proteins.A well-known example is insulin, the hormone regulating glucose metabolismin animals. Insulin is a relatively small protein (5.7 kD) and consists of twopolypeptide chains held together by disulfide cross-bridges. Other hormonesthat are also proteins include pituitary somatotropin (21 kD) and thyrotropin(28 kD), which stimulates the thyroid gland. Another group of regulatory pro-teins is involved in the regulation of gene expression. These proteins charac-teristically act by binding to DNA sequences that are adjacent to coding regionsof genes, either activating or inhibiting the transcription of genetic informa-tion into RNA. Examples include repressors, which, because they block tran-scription, are considered negative control elements.A prokaryotic representative is lac repressor (37 kD), which controls expres-sion of the enzyme system responsible for the metabolism of lactose (milksugar); a mammalian example is NF1 (nuclear factor 1, 60 kD), which inhibitstranscription of the gene encoding the �-globin polypeptide chain of hemo-globin. Positively acting control elements are also known. For example, the E.coli catabolite gene activator protein (CAP) (44 kD), under appropriate meta-bolic conditions, can bind to specific sites along the E. coli chromosome andincrease the rate of transcription of adjacent genes. The mammalian AP1 is aheterodimeric transcription factor composed of one polypeptide from the Junfamily of gene-regulatory proteins and one polypeptide from the Fos family ofgene-regulatory proteins. AP1 activates expression of the �-globin gene. Thesevarious DNA-binding regulatory proteins often possess characteristic structuralfeatures, such as helix-turn-helix, leucine zipper, and zinc finger motifs (seeChapter 31).Transport ProteinsA third class of proteins is the transport proteins. These proteins function totransport specific substances from one place to another. One type of transportis exemplified by the transport of oxygen from the lungs to the tissues by hemo-globin (Figure 5.13a) or by the transport of fatty acids from adipose tissue tovarious organs by the blood protein serum albumin. A very different type is thetransport of metabolites across permeability barriers such as cell membranes,as mediated by specific membrane proteins. These membrane transport proteinstake up metabolite molecules on one side of a membrane, transport themacross the membrane, and release them on the other side. Examples includethe transport proteins responsible for the uptake of essential nutrients into thecell, such as glucose or amino acids (Figure 5.13b). All naturally occurringmembrane transport proteins studied thus far form channels in the membranethrough which the transported substances are passed.Storage ProteinsProteins whose biological function is to provide a reservoir of an essential nutri-ent are called storage proteins. Because proteins are amino acid polymers andbecause nitrogen is commonly a limiting nutrient for growth, organisms haveexploited proteins as a means to provide sufficient nitrogen in times of need.For example, ovalbumin, the protein of egg white, provides the developing birdembryo with a source of nitrogen during its isolation within the egg. Casein isthe most abundant protein of milk and thus the major nitrogen source for122 Chapter 5 ● Proteins: Their Biological Functions and Primary Structuremammalian infants. The seeds of higher plants often contain as much as 60%storage protein to make the germinating seed nitrogen-sufficient during thiscrucial period of plant development. In corn (Zea mays or maize), a family oflow molecular weight proteins in the kernel called zeins serve this purpose; peas(the seeds of Phaseolus vulgaris) contain a storage protein called phaseolin. Theuse of proteins as a reservoir of nitrogen is more efficient than storing an equiv-alent amount of amino acids. Not only is the osmotic pressure minimized, butthe solvent capacity of the cell is taxed less in solvating one molecule of apolypeptide than in dissolving, for example, 100 molecules of free amino acids.Proteins can also serve to store nutrients other than the more obvious elementscomposing amino acids (N, C, H, O, and S). As an example, ferritin is a pro-tein found in animal tissues that binds iron, retaining this essential metal soFIGURE 5.13 ● Two basic types of biologicaltransport are (a) transport within or betweendifferent cells or tissues and (b) transport intoor out of cells. Proteins function in both ofthese phenomena. For example, the proteinhemoglobin transports oxygen from the lungsto actively respiring tissues. Transport proteinsof the other type are localized in cellular mem-branes, where they function in the uptake ofspecific nutrients, such as glucose (shown here)and amino acids, or the export of metabolitesand waste products.5.3 ● The Many Biological Functions of Proteins 123Hemoglobin(Hb) 4 O2Hb(O2)4Arterial circulationVenous circulationLungsHeartTissue(a)Hemoglobin(Hb)4 O2Hb(O2)4(b)Outside InsideGlucoseGlucose transporter(a membrane protein)Cell membranethat it is available for the synthesis of important iron-containing proteins suchas hemoglobin. One molecule of ferritin (460 kD) binds as many as 4500 atomsof iron (35% by weight).Contractile and Motile ProteinsCertain proteins endow cells with unique capabilities for movement. Cell divi-sion, muscle contraction, and cell motility represent some of the ways in whichcells execute motion. The contractile and motile proteins underlying thesemotions share a common property: they are filamentous orpolymerize to formfilaments. Examples include actin and myosin, the filamentous proteins form-ing the contractile systems of cells, and tubulin, the major component of micro-tubules (the filaments involved in the mitotic spindle of cell division as well asin flagella and cilia). Another class of proteins involved in movement includesdynein and kinesin, so-called motor proteins that drive the movement of vesi-cles, granules, and organelles along microtubules serving as establishedcytoskeletal “tracks.”Structural ProteinsAn apparently passive but very important role of proteins is their function increating and maintaining biological structures. Structural proteins providestrength and protection to cells and tissues. Monomeric units of structural pro-teins typically polymerize to generate long fibers (as in hair) or protective sheetsof fibrous arrays, as in cowhide (leather). �-Keratins are insoluble fibrous pro-teins making up hair, horns, and fingernails. Collagen, another insoluble fibrousprotein, is found in bone, connective tissue, tendons, cartilage, and hide, whereit forms inelastic fibrils of great strength. One-third of the total protein in avertebrate animal is collagen. A structural protein having elastic properties is,appropriately, elastin, an important component of ligaments. Because of theway elastin monomers are cross-linked in forming polymers, elastin can stretchin two dimensions. Certain insects make a structurally useful protein, fibroin (a�-keratin), the major constituent of cocoons (silk) and spider webs. An impor-tant protective barrier for animal cells is the extracellular matrix containingcollagen and proteoglycans, covalent protein–polysaccharide complexes that cush-ion and lubricate.Scaffold Proteins (Adapter Proteins)Some proteins play a recently discovered role in the complex pathways of cel-lular response to hormones and growth factors. These proteins, the scaffoldor adapter proteins, have a modular organization in which specific parts (mod-ules) of the protein’s structure recognize and bind certain structural elementsin other proteins through protein–protein interactions. For example, SH2 mod-ules bind to proteins in which a tyrosine residue has become phosphorylatedon its phenolic OOH, and SH3 modules bind to proteins having a character-istic grouping of proline residues. Others include PH modules, which bind tomembranes, and PDZ-containing proteins, which bind specifically to the C-ter-minal amino acid of certain proteins. Because scaffold proteins typically pos-sess several of these different kinds of modules, they can act as a scaffold ontowhich a set of different proteins is assembled into a multiprotein complex.Such assemblages are typically involved in coordinating and communicatingthe many intracellular responses to hormones or other signalling molecules(Figure 5.14). Anchoring (or targeting) proteins are proteins that bind otherproteins, causing them to associate with other structures in the cell. A familyof anchoring proteins, known as AKAP or A kinase anchoring proteins, exists in124 Chapter 5 ● Proteins: Their Biological Functions and Primary Structurewhich specific AKAP members bind the regulatory enzyme protein kinase A(PKA) to particular subcellular compartments. For example, AKAP100 targetsPKA to the endoplasmic reticulum, whereas AKAP79 targets PKA to the plasmamembrane.Protective and Exploitive ProteinsIn contrast to the passive protective nature of some structural proteins, anothergroup can be more aptly classified as protective or exploitive proteins becauseof their biologically active role in cell defense, protection, or exploitation.Prominent among the protective proteins are the immunoglobulins or antibodiesproduced by the lymphocytes of vertebrates. Antibodies have the remarkableability to “ignore” molecules that are an intrinsic part of the host organism, yetthey can specifically recognize and neutralize “foreign” molecules resultingfrom the invasion of the organism by bacteria, viruses, or other infectiousagents. Another group of protective proteins is the blood-clotting proteins,thrombin and fibrinogen, which prevent the loss of blood when the circulatorysystem is damaged. Arctic and Antarctic fishes have antifreeze proteins to protecttheir blood against freezing in the below-zero temperatures of high-latitudeseas. In addition, various proteins serve defensive or exploitive roles for organ-isms, including the lytic and neurotoxic proteins of snake and bee venoms andtoxic plant proteins, such as ricin, whose apparent purpose is to thwart preda-tion by herbivores. Another class of exploitive proteins includes the toxins pro-duced by bacteria, such as diphtheria toxin and cholera toxin.Exotic ProteinsSome proteins display rather exotic functions that do not quite fit the previ-ous classifications. Monellin, a protein found in an African plant, has a verysweet taste and is being considered as an artificial sweetener for human con-sumption. Resilin, a protein having exceptional elastic properties, is found inFIGURE 5.14 ● Diagram of the NnCsequence organization of the adapter proteininsulin receptor substrate-1 (IRS-1) showing thevarious amino acid sequences (in one-lettercode) that contain tyrosine (Y) residues thatare potential sites for phosphorylation. Theother adapter proteins that recognize variousof these sites are shown as Grb2, SHPTP-2, andp85�PIK. Insulin binding to the insulin receptoractivates the enzymatic activity that phosphory-lates these Tyr residues on IRS-1. (Adapted fromWhite, M. F., and Kahn, C. R., 1994. Journal of BiologicalChemistry 269:1–4.)5.3 ● The Many Biological Functions of Proteins 125EY46Y47EY426GSSPENEECY745Y746EY895VNIESY999VDTSNY1172IDLDTY1222ASINSY1010ADMRDY987MTMQEY939MNMDDY727MNMSGY658MMMSDY628MPMSGY608MPMSEY546TEMMNY460ICMGGPEWY107QALL SY578PEEGSY147DTGN Cp85αPIK137156ATP Binding SiteHomology Domainp85αPIKGRB2SHPTP-2the hinges of insect wings. Certain marine organisms such as mussels secreteglue proteins, allowing them to attach firmly to hard surfaces. It is worth repeat-ing that the great diversity of function in proteins, as reflected in this survey,is attained using just 20 amino acids.5.4 ● Some Proteins Have Chemical Groups Other Than Amino AcidsMany proteins consist of only amino acids and contain no other chemicalgroups. The enzyme ribonuclease and the contractile protein actin are twosuch examples. Such proteins are called simple proteins. However, many otherproteins contain various chemical constituents as an integral part of their struc-ture. These proteins are termed conjugated proteins (Table 5.4). If the non-protein part is crucial to the protein’s function, it is referred to as a prostheticgroup. If the nonprotein moiety is not covalently linked to the protein, it canusually be removed by denaturing the protein structure. However, if the con-jugate is covalently joined to the protein, it may be necessary to carry out acidhydrolysis of the protein into its component amino acids in order to release it.Conjugated proteins are typically classified according to the chemical natureof their nonamino acid component; a representative selection of them is givenhere and in Table 5.4. (Note that comparisons of Tables 5.3 and 5.4 reveal twodistinctly different ways of considering the nature of proteins—function ver-sus chemistry.)GLYCOPROTEINS. Glycoproteins are proteins that contain carbohydrate.Proteins destined for an extracellular location are characteristically glycopro-teins. For example, fibronectin and proteoglycans are important componentsof the extracellularmatrix that surrounds the cells of most tissues in animals.Immunoglobulin G molecules are the principal antibody species found circu-lating free in the blood plasma. Many membrane proteins are glycosylated ontheir extracellular segments.LIPOPROTEINS. Blood plasma lipoproteins are prominent examples of theclass of proteins conjugated with lipid. The plasma lipoproteins function pri-marily in the transport of lipids to sites of active membrane synthesis. Serumlevels of low density lipoproteins (LDLs) are often used as a clinical index of sus-ceptibility to vascular disease.NUCLEOPROTEINS. Nucleoprotein conjugates have many roles in the storageand transmission of genetic information. Ribosomes are the sites of proteinsynthesis. Virus particles and even chromosomes are protein–nucleic acid com-plexes.PHOSPHOPROTEINS. These proteins have phosphate groups esterified to thehydroxyls of serine, threonine, or tyrosine residues. Casein, the major proteinof milk, contains many phosphates and serves to bring essential phosphorusto the growing infant. Many key steps in metabolism are regulated betweenstates of activity or inactivity, depending on the presence or absence of phos-phate groups on proteins, as we shall see in Chapter 15. Glycogen phospho-rylase a is one well-studied example.METALLOPROTEINS. Metalloproteins are either metal storage forms, as in thecase of ferritin, or enzymes in which the metal atom participates in a catalyti-cally important manner. We encounter many examples throughout this bookof the vital metabolic functions served by metalloenzymes.126 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureHEMOPROTEINS. These proteins are actually a subclass of metalloproteinsbecause their prosthetic group is heme, the name given to iron protoporphyrinIX (Figure 5.15). Because heme-containing proteins enjoy so many prominentbiological functions, they are considered a class by themselves.FLAVOPROTEINS. Flavin is an essential substance for the activity of a numberof important oxidoreductases. We discuss the chemistry of flavin and its deriv-atives, FMN and FAD, in the chapter on electron transport and oxidative phos-phorylation (Chapter 21).5.4 ● Some Proteins Have Chemical Groups Other Than Amino Acids 127Table 5.4Representative Conjugated ProteinsClass Prosthetic Group Percent by Weight (approx.)Glycoproteins contain carbohydrateFibronectin�-GlobulinProteoglycanLipoproteins contain lipidBlood plasma lipoproteins:High density lipoprotein (HDL) (�-lipoprotein) Triacylglycerols, phospholipids, cholesterol 75Low density lipoprotein (LDL) (�-lipoprotein) Triacylglycerols, phospholipids, cholesterol 67Nucleoprotein complexes contain nucleic acidRibosomes RNA 60Tobacco mosaic virus RNA 5Adenovirus DNAHIV-1 (AIDS virus) RNAPhosphoproteins contain phosphateCasein Phosphate groupsGlycogen phosphorylase a Phosphate groupsMetalloproteins contain metal atomsFerritin Iron 35Alcohol dehydrogenase ZincCytochrome oxidase Copper and ironNitrogenase Molybdenum and ironPyruvate carboxylase ManganeseHemoproteins contain hemeHemoglobinCytochrome cCatalaseNitrate reductaseAmmonium oxidaseFlavoproteins contain flavinSuccinate dehydrogenase FADNADH dehydrogenase FMNDihydroorotate dehydrogenase FAD and FMNSulfite reductase FAD and FMN5.5 ● Reactions of Peptides and ProteinsThe chemical properties of peptides and proteins are most easily consideredin terms of the chemistry of their component functional groups. That is, theypossess reactive amino and carboxyl termini and they display reactions char-acteristic of the chemistry of the R groups of their component amino acids.These reactions are familiar to us from Chapter 4 and from the study of organicchemistry and need not be repeated here.5.6 ● Purification of Protein MixturesCells contain thousands of different proteins. A major problem for proteinchemists is to purify a chosen protein so that they can study its specific prop-erties in the absence of other proteins. Proteins have been separated and puri-fied on the basis of their two prominent physical properties: size and electri-cal charge. A more direct approach is to employ affinity purification strategiesthat take advantage of the biological function or similar specific recognitionproperties of a protein (see Table 5.5 and Chapter Appendix).Separation MethodsSeparation methods based on size include size exclusion chromatography, ultra-filtration, and ultracentrifugation (see Chapter Appendix). The ionic proper-ties of peptides and proteins are determined principally by their complementof amino acid side chains. Furthermore, the ionization of these groups is pH-dependent.A variety of procedures exploit electrical charge as a means of discrimi-nating between proteins, including ion exchange chromatography (seeChapter 4), electrophoresis (see Chapter Appendix), and solubility. Proteinstend to be least soluble at their isoelectric point, the pH value at which thesum of their positive and negative electrical charges is zero. At this pH, elec-trostatic repulsion between protein molecules is minimal and they are morelikely to coalesce and precipitate out of solution. Ionic strength also profoundlyinfluences protein solubility. Most globular proteins tend to become increas-FIGURE 5.15 ● Heme consists of protopor-phyrin IX and an iron atom. Protoporphyrin, ahighly conjugated system of double bonds, iscomposed of four 5-membered heterocyclicrings (pyrroles) fused together to form atetrapyrrole macrocycle. The specific isomericarrangement of methyl, vinyl, and propionateside chains shown is protoporphyrin IX.Coordination of an atom of ferrous iron (Fe2)by the four pyrrole nitrogen atoms yieldsheme.128 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureFe2+–OOCCH2CCH3CHCC CCCOO–H2CCH2CCH3C NHHCC NCCCCH2CCCCHNCCCNCHHCCH3 CHCH2CH3Protoporphyrin IXCCH3CHCC CCCCH3C NHCC NCCCCCCNCCCNCHHCCH3CH3Heme(Fe-protoporphyrin IX)CH2H–OOCCH2CH2COO–H2CCH2CHCH2CH2CHingly soluble as the ionic strength is raised. This phenomenon, the salting-inof proteins, is attributed to the diminishment by the salt ions of electrostaticattractions between the protein molecules. Such electrostatic interactionswould otherwise lead to precipitation. However, as the salt concentrationreaches high levels (greater than 1 M), the effect may reverse so that the pro-tein is salted out of solution. In such cases, the numerous salt ions begin tocompete with the protein for waters of solvation, and, as they win out, the pro-tein becomes insoluble. The solubility properties of a typical protein are shownin Figure 5.16.Although the side chains of most nonpolar amino acids in soluble proteinsare usually buried in the interior of the protein away from contact with theaqueous solvent, a portion of them is exposed at the protein’s surface, giving5.6 ● Purification of Protein Mixtures 129FIGURE 5.16 ● The solubility of most globular proteins is markedly influenced by pHand ionic strength. This figure shows the solubility of a typical protein as a function of pHand various salt concentrations.4.8pH30Solubility, mg of protein/milliliter215.0 5.2 5.4 5.6 5.820 mM10 mM5 mM1 mM4 MA D E E P E R L O O KEstimation of Protein Concentrations in Solutions of Biological OriginBiochemists are often interested in knowing the protein concen-tration in various preparationsof biological origin. Such quanti-tative analysis is not straightforward. Cell extracts are complexmixtures that typically contain protein molecules of many differ-ent molecular weights, so the results of protein estimations can-not be expressed on a molar basis. Also, aside from the ratherunreactive repeating peptide backbone, little common chemicalidentity is seen among the many proteins found in cells that mightbe readily exploited for exact chemical analysis. Most of theirchemical properties vary with their amino acid composition, forexample, nitrogen or sulfur content or the presence of aromatic,hydroxyl, or other functional groups.Lowry ProcedureA method that has been the standard of choice for many years isthe Lowry procedure. This method uses Cu2 ions along withFolin–Ciocalteau reagent, a combination of phosphomolybdic andphosphotungstic acid complexes that react with Cu. Cu is gen-erated from Cu2 by readily oxidizable protein components, suchas cysteine or the phenols and indoles of tyrosine and tryptophan.Although the precise chemistry of the Lowry method remainsuncertain, the Cu reaction with the Folin reagent gives intenselycolored products measurable spectrophotometrically.BCA MethodRecently, a reagent that reacts more efficiently with Cu thanFolin–Ciocalteau reagent has been developed for protein assays.Bicinchoninic acid (BCA) forms a purple complex with Cu in alka-line solution.Assays Based on Dye BindingSeveral other protocols for protein estimation enjoy prevalentusage in biochemical laboratories. The Bradford assay is a rapidand reliable technique that uses a dye called Coomassie BrilliantBlue G-250, which undergoes a change in its color upon non-covalent binding to proteins. The binding is quantitative and lesssensitive to variations in the protein’s amino acid composition.The color change is easily measured by a spectrophotometer. Asimilar, very sensitive method capable of quantifying nanogramamounts of protein is based on the shift in color of colloidal goldupon binding to proteins.Cu BCA 88n �OOC N COO�CuBCA–Cu complex�OOCNN N COO�it a partially hydrophobic character. Hydrophobic interaction chromatography is aprotein purification technique that exploits this hydrophobicity (see ChapterAppendix).A Typical Protein Purification SchemeMost purification procedures for a particular protein are developed in anempirical manner, the overriding principle being purification of the proteinto a homogeneous state with acceptable yield. Table 5.5 presents a summaryof a purification scheme for a selected protein. Note that the specific activityof the protein (the enzyme xanthine dehydrogenase) in the immuno-affinitypurified fraction (fraction 5) has been increased 152/0.108, or 1407 times thespecific activity in the crude extract (fraction 1). Thus, xanthine dehydroge-nase in fraction 5 versus fraction 1 is enriched more than 1400-fold by thepurification procedure.5.7 ● The Primary Structure of a Protein: Determining the Amino Acid SequenceIn 1953, Frederick Sanger of Cambridge University in England reported theamino acid sequences of the two polypeptide chains composing the proteininsulin (Figure 5.17). Not only was this a remarkable achievement in analyti-cal chemistry but it helped to demystify speculation about the chemical natureof proteins. Sanger’s results clearly established that all of the molecules of agiven protein have a fixed amino acid composition, a defined amino acidsequence, and therefore an invariant molecular weight. In short, proteins arewell defined chemically. Today, the amino acid sequences of some 100,000 dif-ferent proteins are known. Although many sequences have been determinedfrom application of the principles first established by Sanger, most are nowdeduced from knowledge of the nucleotide sequence of the gene that encodesthe protein.130 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureTable 5.5Example of a Protein Purification Scheme: Purification of the Enzyme Xanthine Dehydrogenase from a FungusVolume Total Total Specific PercentFraction (mL) Protein (mg) Activity* Activity† Recovery‡1. Crude extract 3,800 22,800 2,460 0.108 1002. Salt precipitate ,165 2,800 1,190 0.425 483. Ion exchange chromatography ,65 ,100 ,720 7.2 294. Molecular sieve chromatography ,40 ,14.5 ,555 38.3 235. Immunoaffinity chromatography§ ,6 1.8 ,275 152, 11*The relative enzymatic activity of each fraction in catalyzing the xanthine dehydrogenase reaction is cited as arbitrarily defined units.†The specific activity is the total activity of the fraction divided by the total protein in the fraction. This value gives an indication of the increase inpurity attained during the course of the purification as the samples become enriched for xanthine dehydrogenase protein.‡The percent recovery of total activity is a measure of the yield of the desired product, xanthine dehydrogenase.§The last step in the procedure is an affinity method in which antibodies specific for xanthine dehydrogenase are covalently coupled to a chromatog-raphy matrix and packed into a glass tube to make a chromatographic column through which fraction 4 is passed. The enzyme is bound by thisimmunoaffinity matrix while other proteins pass freely out. The enzyme is then recovered by passing a strong salt solution through the column, whichdissociates the enzyme–antibody complex.Adapted from Lyon, E. S., and Garrett, R. H., 1978. Journal of Biological Chemistry. 253:2604–2614.Protein Sequencing StrategyThe usual strategy for determining the amino acid sequence of a proteininvolves eight basic steps:1. If the protein contains more than one polypeptide chain, the chains areseparated and purified.2. Intrachain SOS (disulfide) cross-bridges between cysteine residues in thepolypeptide chain are cleaved. (If these disulfides are interchain linkages,then step 2 precedes step 1.)3. The amino acid composition of each polypeptide chain is determined.4. The N-terminal and C-terminal residues are identified.5. Each polypeptide chain is cleaved into smaller fragments, and the aminoacid composition and sequence of each fragment are determined.6. Step 5 is repeated, using a different cleavage procedure to generate a dif-ferent and therefore overlapping set of peptide fragments.7. The overall amino acid sequence of the protein is reconstructed from thesequences in overlapping fragments.8. The positions of SOS cross-bridges formed between cysteine residues arelocated.Each of these steps is discussed in greater detail in the following sections.Step 1. Separation of Polypeptide ChainsIf the protein of interest is a heteromultimer (composed of more than one typeof polypeptide chain), then the protein must be dissociated and its componentpolypeptide subunits must be separated from one another and sequenced indi-vidually. Subunit associations in multimeric proteins are typically maintainedsolely by noncovalent forces, and therefore most multimeric proteins can usu-ally be dissociated by exposure to pH extremes, 8 M urea, 6 M guanidiniumhydrochloride, or high salt concentrations. (All of these treatments disruptpolar interactions such as hydrogen bonds both within the protein moleculeand between the protein and the aqueous solvent.) Once dissociated, the indi-vidual polypeptides can be isolated from one another on the basis of differ-ences in size and/or charge. Occasionally, heteromultimers are linked togetherby interchain SOS bridges. In such instances, these cross-links must be cleavedprior to dissociation and isolation of the individual chains. The methodsdescribed under step 2 are applicable for this purpose.Step 2. Cleavage of Disulfide BridgesA number of methods exist for cleavingdisulfides (Figure 5.18). An importantconsideration is to carry out these cleavages so that the original or even newSOS links do not form. Oxidation of a disulfide by performic acid results inthe formation of two equivalents of cysteic acid (Figure 5.18a). Because thesecysteic acid side chains are ionized SO3� groups, electrostatic repulsion (as wellas altered chemistry) prevents SOS recombination. Alternatively, sulfhydrylcompounds such as 2-mercaptoethanol readily reduce SOS bridges to regen-erate two cysteineOSH side chains (Figure 5.18b). However, these SH groupsrecombine to re-form either the original disulfide link or, if other free CysOSHsare available, new disulfide links. To prevent this, SOS reduction must be fol-lowed by treatment with alkylating agents such as iodoacetate or 3-bromo-propylamine, which modify the SH groups and block disulfide bridge forma-tion (Figure 5.18a).FIGURE 5.17 ● The hormone insulin consistsof two polypeptide chains, A and B, heldtogether by two disulfide cross-bridges (SOS).The A chain has 21 amino acid residues and anintrachain disulfide; the B polypeptide contains30 amino acids. The sequence shown is forbovine insulin.5.7 ● The Primary Structure of a Protein: Determining the Amino Acid Sequence 131S SGlyIleValGluGlnCysCysAlaSerValCysSerLeuTyrGlnLeuGluAsnTyrCysAsnPheValAsnGlnHisLeuCysGlySerHisLeuValGluAlaLeuTyrLeuValCysGlyGluArgGlyPhePheTyrThrProLysAla52015103025S SB chainA chainSSN NCCStep 3. Analysis of Amino Acid CompositionThe standard protocol for analysis of the amino acid composition of proteinsis discussed in Section 5.1. Results of such analyses allow the researcher to antic-ipate which methods of polypeptide fragmentation might be useful for the protein.Step 4. Identification of the N- and C-Terminal ResiduesEnd-group analysis reveals several things. First, it identifies the N- and C-ter-minal residues in the polypeptide chain. Second, it can be a clue to the num-ber of ends in the protein. That is, if the protein consists of two or more dif-ferent polypeptide chains, then more than one end group may be discovered,alerting the investigator to the presence of multiple polypeptides.FIGURE 5.18 ● Methods for cleavage ofdisulfide bonds in proteins. (a) Oxidative cleav-age by reaction with performic acid. (b) Re-ductive cleavage with sulfhydryl compounds.Disulfide bridges can be broken by reductionof the SOS link with sulfhydryl agents such as2-mercaptoethanol or dithiothreitol. Becausereaction between the newly reduced OSHgroups to re-establish disulfide bonds is a likeli-hood, SOS reduction must be followed byOSH modification: (1) alkylation with iodoac-etate (ICH2COOH) or (2) modification with 3-bromopropylamine (BrO(CH2)3ONH2).132SDisulfidebond...+(a)N CH CRHCH2ON CH CHOCH2NH...SSCH2... N CH CR'HON CH CHONH...Cysteic acidresidues... N CH CRHON CH CHOCH2NH...SO3–CH2... N CH CR'HON CH CHONH...SO3–H COO O HPerformic acid...(b)N C CHHOCH2SSCH2C...... N CHO...H2 HSCH2CH2OH2-mercaptoethanol+... N C CHHOCH2SHSHCH2C...... N CHO...HSSCH2 OHCH2 CH2 OH(1)... N C CHHOCH2SH... + ICH2COOHIodoacetic acid3-BromopropylamineHI ++... N C CHHOCH2S...CH2 COO–S-carboxymethyl derivative(2)... N C CHHOCH2... + ... N C CHHOCH2...CH2 CH2CH2 NH2SHCH2Br HBrCH2CH2NH2A. N-Terminal AnalysisThe amino acid residing at the N-terminal end of a protein can be identifiedin a number of ways; one method, Edman degradation, has become the pro-cedure of choice. This method is preferable because it allows the sequentialidentification of a series of residues beginning at the N-terminus (Figure 5.19).In weakly basic solutions, phenylisothiocyanate, or Edman’s reagent(phenylONPCPS), combines with the free amino terminus of a protein(Figure 5.19), which can be excised from the end of the polypeptide chain andrecovered as a phenylthiohydantoin (PTH) derivative. This PTH derivative canbe identified by chromatographic methods. Importantly, in this procedure, therest of the polypeptide chain remains intact and can be subjected to furtherrounds of Edman degradation to identify successive amino acid residues in thechain. Often, the carboxyl terminus of the polypeptide under analysis is cou-pled to an insoluble matrix, allowing the polypeptide to be easily recovered byfiltration following each round of Edman reaction. Thus, Edman reaction notonly identifies the N-terminus of proteins but can also reveal additional infor-mation regarding sequence. Automated instruments (so-called Edman seque-nators) have been designed to carry out the reaction cycle of the Edman pro-cedure. In practical terms, as many as 50 cycles of reaction can be accomplished5.7 ● The Primary Structure of a Protein: Determining the Amino Acid Sequence 133FIGURE 5.19 ● N-Terminal analysis using Edman’s reagent, phenylisothiocyanate.Phenylisothiocyanate combines with the N-terminus of a peptide under mildly alkalineconditions to form a phenylthiocarbamoyl substitution. Upon treatment with TFA (trifluo-roacetic acid), this cyclizes to release the N-terminal amino acid residue as a thiazolinonederivative, but the other peptide bonds are not hydrolyzed. Organic extraction and treat-ment with aqueous acid yield the N-terminal amino acid as a phenylthiohydantoin (PTH)derivative.NH2CHCNC OH NR' CHOH NR'' CHC OR...+NCSMild alkaliCHCC OH NOH NC O...NCNTFAWeakaqueous acidThiazolinonederivativeHSHRCHR'CHR''CH CRNHCSN NCOOS CR HPTH-derivativePeptide chainone residueshorterPeptide chainNCHCC OH NO...CHR''H3NCHR'+on 50 pmol (about 0.1 g) of a polypeptide 100 to 200 residues long, gener-ating the sequential order of the first 50 amino acid residues in the protein.The efficiency with larger proteins is less; a typical 2000-amino acid proteinprovides only 10 to 20 cycles of reaction.B. C-Terminal AnalysisFor the identification of the C-terminal residue of polypeptides, an enzymaticapproach is commonly used.ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are en-zymes that cleave amino acid residues from the C-termini of polypeptides in asuccessive fashion. Four carboxypeptidases are in general use: A, B, C, and Y.Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. Theanalogous enzyme from hog pancreas, carboxypeptidase B, is effective only whenArg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidasesA and B liberates any C-terminal amino acid except proline. Carboxypeptidase Cfrom citrus leaves and carboxypeptidase Y from yeast act on any C-terminalresidue. Because the nature of the amino acid residue at the end often deter-mines the rate at which it is cleaved and because these enzymes remove residuessuccessively, care must be taken in interpretingresults. Carboxypeptidase Ycleavage has been adapted to an automated protocol analogous to that usedin Edman sequenators.Steps 5 and 6. Fragmentation of the Polypeptide ChainThe aim at this step is to produce fragments useful for sequence analysis. Thecleavage methods employed are usually enzymatic, but proteins can also befragmented by specific or nonspecific chemical means (such as partial acidhydrolysis). Proteolytic enzymes offer an advantage in that they may hydrolyzeonly specific peptide bonds, and this specificity immediately gives informationabout the peptide products. As a first approximation, fragments produced uponcleavage should be small enough to yield their sequences through end-groupanalysis and Edman degradation, yet not so small that an over-abundance ofproducts must be resolved before analysis. However, the determination of totalsequences for proteins predates the Edman procedure, and alternativeapproaches obviously exist.A. TrypsinThe digestive enzyme trypsin is the most commonly used reagent for specificproteolysis. Trypsin is specific in hydrolyzing only peptide bonds in which thecarbonyl function is contributed by an arginine or a lysine residue. That is,trypsin cleaves on the C-side of Arg or Lys, generating a set of peptide frag-ments having Arg or Lys at their C-termini. The number of smaller peptidesresulting from trypsin action is equal to the total number of Arg and Lysresidues in the protein plus one—the protein’s C-terminal peptide fragment(Figure 5.20).B. ChymotrypsinChymotrypsin shows a strong preference for hydrolyzing peptide bonds formedby the carboxyl groups of the aromatic amino acids, phenylalanine, tyrosine,and tryptophan. However, over time chymotrypsin also hydrolyzes amide bondsinvolving amino acids other than Phe, Tyr, or Trp. Peptide bonds havingleucine-donated carboxyls become particularly susceptible. Thus, the specificity134 Chapter 5 ● Proteins: Their Biological Functions and Primary Structureof chymotrypsin is only relative. Because chymotrypsin produces a very differ-ent set of products than trypsin, treatment of separate samples of a proteinwith these two enzymes generates fragments whose sequences overlap.Resolution of the order of amino acid residues in the fragments yields theamino acid sequence in the original protein.C. Relatively Nonspecific EndopeptidasesA number of other endopeptidases (proteases that cleave peptide bonds withinthe interior of a polypeptide chain) are also used in sequence investigations.These include clostripain, which acts only at Arg residues, endopeptidase Lys-C,which cleaves only at Lys residues, and staphylococcal protease, which acts at theacidic residues, Asp and Glu. Other, relatively nonspecific endopeptidases arehandy for digesting large tryptic or chymotryptic fragments. Pepsin, papain, sub-tilisin, thermolysin, and elastase are some examples. Papain is the active ingredi-ent in meat tenderizer and in soft contact lens cleaner as well as in some laun-dry detergents. The abundance of papain in papaya, and a similar protease(bromelain) in pineapple, causes the hydrolysis of gelatin and prevents thepreparation of Jell-O® containing either of these fresh fruits. Cooking thesefruits thermally denatures their proteolytic enzymes so that they can be usedin gelatin desserts.D. Cyanogen BromideSeveral highly specific chemical methods of proteolysis are available, the mostwidely used being cyanogen bromide (CNBr) cleavage. CNBr acts upon methio-FIGURE 5.20 ● Trypsin is a proteolyticenzyme, or protease, that specifically cleaves onlythose peptide bonds in which arginine or lysinecontributes the carbonyl function. The prod-ucts of the reaction are a mixture of peptidefragments with C-terminal Arg or Lys residuesand a single peptide derived from the polypep-tide’s C-terminal end.5.7 ● The Primary Structure of a Protein: Determining the Amino Acid Sequence 135(b)N—Asp—Ala—Gly—Arg—His—Cys—Lys—Trp—Lys—Ser—Glu—Asn—Leu—Ile—Arg—Thr—Tyr—CTrypsinAsp—Ala—Gly—ArgHis—Cys—LysTrp—LysSer—Glu—Asn—Leu—Ile—ArgThr—TyrNHCH C CHO CH2CH2CH2HNC NH2NH2NHCH3+CONHCHCH2OHCONHCHCH2CH2CH2CH2NH3+CONHCHCH2COO–CO...(a)TrypsinAlaTrypsin...Arg Ser Lys Aspnine residues (Figure 5.21). The nucleophilic sulfur atom of Met reacts withCNBr, yielding a sulfonium ion that undergoes a rapid intramolecularrearrangement to form a cyclic iminolactone. Water readily hydrolyzes this imi-nolactone, cleaving the polypeptide and generating peptide fragments havingC-terminal homoserine lactone residues at the former Met positions.Other Methods of FragmentationA number of other chemical methods give specific fragmentation of polypep-tides, including cleavage at asparagine–glycine bonds by hydroxylamine(NH2OH) at pH 9 and selective hydrolysis at aspartyl–prolyl bonds under mildlyacidic conditions. Table 5.6 summarizes the various procedures described herefor polypeptide cleavage. These methods are only a partial list of the arsenalof reactions available to protein chemists. Cleavage products generated by theseprocedures must be isolated and individually analyzed with respect to aminoacid composition, end-group identity, and amino acid sequence to accumulatethe information necessary to reconstruct the protein’s total amino acidsequence. In the past, sequence was often deduced from exhaustive study ofthe amino acid composition and end-group analysis of small, overlapping pep-tides. Peptide sequencing today is most commonly done by Edman degrada-tion of relatively large peptides.Sequence Determination by Mass Spectrometry (MS)Mass spectrometers exploit the difference in the mass-to-charge (m/z) ratio ofionized atoms or molecules to separate them from each other. The m/z ratioof a molecule is also a highly characteristic property that can be used for deter-mining chemical and structural information. Further, molecules can be frag-mented in distinctive ways in mass spectrometers, and the fragments that arisealso provide quite specific structural information about the molecule. The basicFIGURE 5.21 ● Cyanogen bromide (CNBr) isa highly selective reagent for cleavage of pep-tides only at methionine residues. (I) The reac-tion occurs in 70% formic acid via nucleophilicattack of the Met S atom on the OCqN car-bon atom, with displacement of Br. (II) Thecyano intermediate undergoes nucleophilicattack by the Met carbonyl oxygen atom on theR group, (III) resulting in formation of thecyclic derivative, which is unstable in aqueoussolution. (IV) Hydrolysis ensues, producingcleavage of the Met peptide bond and releaseof peptide fragments, with C-terminal homoser-ine lactone residues where Met residues oncewere. One peptide does not have a C-terminalhomoserine lactone: the original C-terminalend of the polypeptide.136 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureSCH3CH2CH2C COHNHNCδ+Brδ–N Br–SCH3CH2CH2COHNHN+I IIH3C S C NCCCH2NNCH2 O+Methyl thiocyanateIIICCCH2ONCH2 OIVCH3CH2SCH2C COHNH CHNOVERALL REACTION:Polypeptide70%HCOOHCH2C CH2OPeptide with C-terminalhomoserine lactoneC N+H2OBrCNHCH H HH H HH... ... ... ... ... ... ............ONH+H3N Peptide(C-terminal peptide)+ Peptide(C-terminal peptide)operation of a mass spectrometer is to (1) evaporate and ionize molecules ina vacuum, creating gas-phaseions, (2) separate the ions in space and/or timebased on their m/z ratios, and (3) measure the amount of ions with specificm/z ratios. Because proteins (as well as nucleic acids and carbohydrates) decom-pose upon heating, rather than evaporating, attempts to ionize such moleculesfor MS analysis require innovative approaches (Table 5.7). Figure 5.22 illus-5.7 ● The Primary Structure of a Protein: Determining the Amino Acid Sequence 137Table 5.6Specificity of Representative Polypeptide Cleavage Procedures Used in Sequence AnalysisPeptide Bond onCarboxyl (C) or Amino (N) SusceptibleMethod Side of Susceptible Residue Residue(s)Proteolytic enzymesTrypsin C Arg or LysChymotrypsin C Phe, Trp, or Tyr; LeuClostripain C ArgStaphylococcal protease C Asp or GluChemical methodsCyanogen bromide C MetNH2OH Asn-Gly bondspH 2.5, 40°C Asp-Pro bondsTable 5.7Macromolecular Ionization Methods in Mass SpectrometryElectrospray ionization (ESI-MS) A solution of macromolecules issprayed in the form of fine dropletsfrom a glass capillary under theinfluence of a strong electrical field.The droplets pick up charge as theyexit the capillary; evaporation of thesolvent leaves highly charged molecules.Fast-atom bombardment A high-energy beam of inert gas(FAB-MS) molecules (argon or xenon) is directed ata solid sample, knocking molecules intothe gas phase and ionizing them.Laser ionization (LIMS) A laser pulse is used to knock material fromthe surface of a solid sample; the laser pulsecreates a microplasma that ionizes moleculesin the sample.Matrix-assisted desorption MALDI is a LIMS method capable ofionization (MALDI) vaporizing and ionizing large biologicalmolecules such as proteins or DNA. Thebiological molecules are dispersed in asolid matrix that serves as a carrier.Nicotinic acid is a commonly used matrixsubstance.138 Chapter 5 ● Proteins: Their Biological Functions and Primary Structure+ +++++++++++ ++++++Mass spectrometer(a) High voltageSamplesolutionGlass capillaryCounter-currentVacuuminterface+(b)(c)2550Intensity (%) 0751001000800 1200 1400 1600m/z4700047342050+5010048000Molecular weight40+30+FIGURE 5.22 ● The three principal steps in electrospray mass spectrometry (ES-MS).(a) Small, highly charged droplets are formed by electrostatic dispersion of a protein solu-tion through a glass capillary subjected to a high electric field; (b) protein ions are de-sorbed from the droplets into the gas phase (assisted by evaporation of the droplets in astream of hot N2 gas; and (c) analysis of the protein ions in a mass spectrometer. (Adapted from Figure 1 in Mann, M., and Wilm, M., 1995. Trends in Biochemical Sciences 20:219–224.)FIGURE 5.23 ● Electrospray mass spectrum of the protein, aerolysin K. The attachment of many protons per proteinmolecule (from less than 30 to more than 50 here) leads to a series of m/z peaks for this single protein. The inset shows acomputer analysis of the data from this series of peaks that generates a single peak at the correct molecular mass of theprotein. (Adapted from Figure 2 in Mann, M., and Wilm, M., 1995. Trends in Biochemical Sciences 20:219–224.)trates the basic features of electrospray mass spectrometry (ES-MS). In this tech-nique, proteins pick up, on average, about one positive charge (proton) perkilodalton, leading to the spectrum of m/z ratios for a single protein species(Figure 5.23). Computer algorithms can convert these data into a single spec-trum having a peak at the correct protein mass (inset, Figure 5.23).SEQUENCING BY TANDEM MASS SPECTROMETRY. Tandem MS (or MS/MS) allowssequencing of proteins by hooking two mass spectrometers in tandem. The firstmass spectrometer is used to separate oligopeptides from a protein digest andthen to select in turn each of these oligopeptides for further analysis. A selectedionized oligopeptide is directed toward the second mass spectrometer; on theway, this oligopeptide is fragmented by collision with helium or argon gas mole-cules, and the collection of fragments is analyzed by the second mass spec-trometer (Figure 5.24). Fragmentation occurs primarily in the peptide bonds5.7 ● The Primary Structure of a Protein: Determining the Amino Acid Sequence 139FIGURE 5.24 ● Tandem mass spectrometry. (a) Configuration used in tandem MS. (b) Schematic description of tandem MS: tandem MS involves electrospray ionization of aprotein digest (IS in this figure), followed by selection of a single peptide ion mass for col-lision with inert gas molecules (He) and mass analysis of the fragment ions resulting fromthe collisions. (c) Fragmentation usually occurs at peptide bonds, as indicated. (I: Adapted from Yates, J. R., 1996. Methods in Enzymology 271:351–376; II: Adapted from Gillece-Castro, B. L., andStults, J. T., 1996. Methods in Enzymology 271:427–447.)Electrospray Ionization Tandem Mass SpectrometerFragmentationat peptidebondsElectrosprayIonizationSourceMS-1 Collision Cell MS-2 DetectorCR1N CHHP1P2P3P4P5F1 F2 F3 F4 F5NHO OCR2CHNHOCR3CHMS-1 MS-2HegasCollisioncellISDetElectrosprayIonization(a)(c)(b)......linking successive amino acids in the oligopeptide, so the fragments createdrepresent a nested set of peptides that differ in size by one amino acid residue.The fragments differ in mass by 56 atomic mass units (the mass of the peptidebackbone atoms (NH-CH-CO)) plus the mass of the R group at each position,which ranges from 1 atomic mass unit (Gly) to 130 (Trp). MS sequencing hasthe advantages of very high sensitivity, fast sample processing, and the abilityto work with mixtures of proteins. Subpicomoles (less than 10�12 moles) ofpeptide can be analyzed. However, in practice, tandem MS is limited to rathershort sequences (no longer than 15 or so amino acid residues). Nevertheless,capillary HPLC-separated peptide mixtures from trypsin digests of proteins canbe directly loaded into the tandem MS spectrometer. Further, separation of acomplex mixture of proteins from a whole-cell extract by two-dimensional gelelectrophoresis (see Chapter Appendix), followed by trypsin digest of a spe-cific protein spot on the gel and injection of the digest into the HPLC/tan-dem MS, gives sequence information that can be used to identify specific pro-teins. Often, by comparing the mass of tryptic peptides from a protein digestwith a database of all possible masses for tryptic peptides (based on all knownprotein and DNA sequences), a protein of interest can be identified withoutactually sequencing it.Step 7. Reconstruction of the Overall Amino Acid SequenceThe sequences obtained for the sets of fragments derived from two or morecleavage procedures are now compared, with the objective being to find over-laps that establish continuity of the overall amino acid sequence of the polypep-tide chain. The strategy is illustrated by the example shown in Figure 5.25.Peptides generated from specific hydrolysis of the polypeptide can be alignedto reveal the overall amino acid sequence. Such comparisons are also useful ineliminating errors and validating the accuracy of the sequences determinedfor the individual fragments.FIGURE 5.25 ● Summary of the sequenceanalysis of catrocollastatin-C, a 23.6-kD proteinfound in the venom of the western diamond-back rattlesnake Crotalus atrox. Sequencesshown are given in the one-letter amino acidcode. The overall amino acid sequence (216amino acid residues long) for catrocollastatin-Cas deduced from the overlapping sequences ofpeptide fragments is shown on the linesheadedCAT-C. The other lines report the vari-ous sequences used to obtain the overlaps.These sequences were obtained from (a) N-term.: Edman degradation of the intact proteinin an automated Edman sequenator; (b) M:proteolytic fragments generated by CNBr cleav-age, followed by Edman sequencing of the indi-vidual fragments (numbers denote fragmentsM1 through M5); (c) K: proteolytic fragments(K3 through K6) from endopeptidase Lys-Ccleavage, followed by Edman sequencing; (d) E: proteolytic fragments from Staphylococcusprotease (E13 through E15) digestion of catro-collastatin sequenced in the Edman sequenator.(Adapted from Shimokawa, K., et al., 1997. Archives ofBiochemistry and Biophysics 343:35–43.)140 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureGSQCGHGDCCEQCKFSKSGTECRASMSECDPAEHCTGQSSECPADVFHKNGQPCLDNYGYCY NGNCPIMYHQCYDLKKSGTECRASMSECDPAEHCTGQSSECPADVFNGQPCLDNYGYCYNGNCPIMYHQCYDLSECDPAEHCTGQSSECPADVFHKNGQPCLDNYGYCYYHQCYDLFGADVYEAEDSCFERNQKGNYYGYCRKENGNKIPCCAPEDVKCGRLYCKDNSPGQNNPCKM–SCFERNQKGNDVKCGRLYCKDNSPGQNNPCKMFGADVYEAEDSCFFGAFYSNEDEHKGMVLPGTKCADGKVCSNGHCVDVATAYFYSNEDEHKGMVLPGTKCADGKVCSNGHCVDVATAYFYSNEDEHKGMVLPGTKCADGKVCCAT-CCAT-CCAT-CCAT-CN-TermM1M1K3K4M2M3M3K4K5K6K6E13E15E15M5M41 10 20 30 40 50 6070 80 90 110100 120130 140 150 160 170 180190 200 210–F––RNQKGNYYGYCRKENGNKIPCCAPEDVKCGRLYCKDN–PGQN– PCKLGTDIISPPVCGNELLEVGEECDCGTPENCQNECCDAATCKLKSGSQCGHGDCCEQCKFSLGTDIISPPVCGNELLEVGEECDCGTPENCQNECCDAATLGTDIISPPVCGNELLEVGEECDCGTPENCQNECCDAATCKLKSGSQCGHGDCCEQCStep 8. Location of Disulfide Cross-BridgesStrictly speaking, the disulfide bonds formed between cysteine residues in aprotein are not a part of its primary structure. Nevertheless, information abouttheir location can be obtained by procedures used in sequencing, provided thedisulfides are not broken prior to cleaving the polypeptide chain. Because thesecovalent bonds are stable under most conditions used in the cleavage ofpolypeptides, intact disulfides link the peptide fragments containing their spe-cific cysteinyl residues and thus these linked fragments can be isolated andidentified within the protein digest.An effective way to isolate these fragments is through diagonal elec-trophoresis (Figure 5.26) (the basic technique of electrophoresis is described inFIGURE 5.26 ● Disulfide bridges typically arecleaved prior to determining the primary struc-ture of a polypeptide. Consequently, the posi-tions of disulfide links are not obvious from thesequence data. To determine their location, asample of the polypeptide with intact SOSbonds can be fragmented and the sites of anydisulfides can be elucidated from fragmentsthat remain linked. Diagonal electrophoresis is atechnique for identifying such fragments. (a) Aprotein digest in which any disulfide bondsremain intact and link their respective Cys-con-taining peptides is streaked along the edge of afilter paper and (b) subjected to electrophore-sis. (c) A strip cut from the edge of the paperis then exposed to performic acid fumes to oxi-dize any disulfide bridges. (d) Then the paperstrip is attached to a new filter paper so that asecond electrophoresis can be run in a direc-tion perpendicular to the first. (e) Peptidesdevoid of disulfides experience no mobilitychange, and thus their pattern of migrationdefines a diagonal. Peptides that had disulfidesmigrate off this diagonal and can be easilyidentified, isolated, and sequenced to revealthe location of cysteic acid residues formerlyinvolved in disulfide bridges.5.7 ● The Primary Structure of a Protein: Determining the Amino Acid Sequence 141++––Partial proteindigest of sample is smeared along oneedge of paper(a)(b)BufferMigration of peptidestoward – electrode(c)(d)HCOOOH-treatedstrip is attachedto new sheet ofpaper and secondelectrophoresis runis performed(e)DiagonalPeptides derivedfrom disulfide-linkedprotein fragmentsSample strip is cut fromelectrophoretogram and treatedwith performic acid vaporsPerformic acidthe Chapter Appendix). Peptides that were originally linked by disulfides nowmigrate as distinct species following disulfide cleavage and are obvious by theirlocation off the diagonal (Figure 5.26e). These cysteic acid–containing pep-tides are then isolated from the paper and sequenced. From this information,the positions of the disulfides in the protein can be stipulated.Sequence DatabasesA database of protein sequences collected by protein chemists can be foundin the Atlas of Protein Sequence and Structure. However, most protein sequenceinformation has been derived from translating the nucleotide sequences ofgenes into codons and, thus, amino acid sequences (see Chapter 13).Sequencing the order of nucleotides in cloned genes is a more rapid, efficient,and informative process than determining the amino acid sequences of pro-teins. A number of electronic databases containing continuously updatedsequence information are readily accessible by personal computer. Prominentamong these are PIR (Protein Identification Resource Protein SequenceDatabase), GenBank (Genetic Sequence Data Bank), and EMBL (EuropeanMolecular Biology Laboratory Data Library).5.8 ● Nature of Amino Acid SequencesWith a knowledge of the methodology in hand, let’s review the results of aminoacid composition and sequence studies on proteins. Table 5.8 lists the relativefrequencies of the amino acids in various proteins. It is very unusual for a glob-ular protein to have an amino acid composition that deviates substantially fromthese values. Apparently, these abundances reflect a distribution of amino acidpolarities that is optimal for protein stability in an aqueous milieu. Membraneproteins have relatively more hydrophobic and fewer ionic amino acids, a con-dition consistent with their location. Fibrous proteins may show compositionsthat are atypical with respect to these norms, indicating an underlying rela-tionship between the composition and the structure of these proteins.Proteins have unique amino acid sequences, and it is this uniqueness ofsequence that ultimately gives each protein its own particular personality.Because the number of possible amino acid sequences in a protein is astro-nomically large, the probability that two proteins will, by chance, have similaramino acid sequences is negligible. Consequently, sequence similaritiesbetween proteins imply evolutionary relatedness.Homologous Proteins from Different Organisms Have Homologous Amino Acid SequencesProteins sharing a significant degree of sequence similarity are said to be homol-ogous. Proteins that perform the same function in different organisms are alsoreferred to as homologous. For example, the oxygen transport protein, hemo-globin, serves a similar role and has a similar structure in all vertebrates. Thestudy of the amino acid sequences of homologous proteins from differentorganisms provides very strong evidence for their evolutionary origin within acommon ancestor. Homologous proteins characteristically have polypeptidechains that are nearly identical in length, and their sequences share identityin direct correlation to the relatedness of the species from which they arederived.142 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureCytochrome cThe electron transport protein, cytochrome c, found in the mitochondria ofall eukaryotic organisms, provides the best-studied example of homology. Thepolypeptide chain of cytochrome c from most species contains slightly morethan 100 amino acids and has a molecular weight of about 12.5 kD. Aminoacid sequencing of cytochromec from more than 40 different species hasrevealed that there are 28 positions in the polypeptide chain where the sameamino acid residues are always found (Figure 5.27). These invariant residuesapparently serve roles crucial to the biological function of this protein, andthus substitutions of other amino acids at these positions cannot be tolerated.FIGURE 5.27 ● Cytochrome c is a small protein consisting of a single polypeptide chainof 104 residues in terrestrial vertebrates, 103 or 104 in fishes, 107 in insects, 107 to 109 infungi and yeasts, and 111 or 112 in green plants. Analysis of the sequence of cytochrome cfrom more than 40 different species reveals that 28 residues are invariant. These invariantresidues are scattered irregularly along the polypeptide chain, except for a cluster betweenresidues 70 and 80. All cytochrome c polypeptide chains have a cysteine residue at position17, and all but one have another Cys at position 14. These Cys residues serve to link theheme prosthetic group of cytochrome c to the protein, a role explaining their invariablepresence.5.8 ● Nature of Amino Acid Sequences 143Table 5.8Frequency of Occurrence of Amino Acid Residues in ProteinsOccurrence inAmino Acid Mr* Proteins (%)†Alanine Ala A 71.1 9.0Arginine Arg R 156.2 4.7Asparagine Asn N 114.1 4.4Aspartic acid Asp D 115.1 5.5Cysteine Cys C 103.1 2.8Glutamine Gln Q 128.1 3.9Glutamic acid Glu E 129.1 6.2Glycine Gly G 57.1 7.5Histidine His H 137.2 2.1Isoleucine Ile I 113.2 4.6Leucine Leu L 113.2 7.5Lysine Lys K 128.2 7.0Methionine Met M 131.2 1.7Phenylalanine Phe F 147.2 3.5Proline Pro P 97.1 4.6Serine Ser S 87.1 7.1Threonine Thr T 101.1 6.0Tryptophan Trp W 186.2 1.1Tyrosine Tyr Y 163.2 3.5Valine Val V 99.1 6.9*Molecular weight of amino acid minus that of water.†Frequency of occurrence of each amino acid residue in the polypeptide chains of 207 unre-lated proteins of known sequence. Values from Klapper, M. H., 1977. Biochemical and Biophysical Research Communications 78:1018–1024.PheCysHisGlyProLeuGlyArgGlyGlyTyrTrpLeuAsnPro LysLysProThrLysMetPheGlyArg171829303234384145485968707176787980828491107273HemeGly1Gly6Asn52Tyr74100▲Furthermore, as shown in Figure 5.28, the number of amino acid differ-ences between two cytochrome c sequences is proportional to the phylogeneticdifference between the species from which they are derived. The cytochromec in humans and in chimpanzees is identical; human and another mammalian(sheep) cytochrome c differ at 10 residues. The human cytochrome c sequencehas 14 variant residues from a reptile sequence (rattlesnake), 18 from a fish(carp), 29 from a mollusc (snail), 31 from an insect (moth), and more than40 from yeast or higher plants (cauliflower).The Phylogenetic Tree for Cytochrome cFigure 5.29 displays a phylogenetic tree (a diagram illustrating the evolution-ary relationships among a group of organisms) constructed from the sequencesof cytochrome c. The tips of the branches are occupied by contemporary specieswhose sequences have been determined. The tree has been deduced by com-puter analysis of these sequences to find the minimum number of mutationalchanges connecting the branches. Other computer methods can be used toinfer potential ancestral sequences represented by nodes, or branch points, inthe tree. Such analysis ultimately suggests a primordial cytochrome c sequencelying at the base of the tree. Evolutionary trees constructed in this manner,that is, solely on the basis of amino acid differences occurring in the primarysequence of one selected protein, show remarkable agreement with phyloge-netic relationships derived from more classic approaches and have given riseto the field of molecular evolution.144 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureFIGURE 5.28 ● The number of amino acid differences among the cytochrome csequences of various organisms can be compared. The numbers bear a direct relationshipto the degree of relatedness between the organisms. Each of these species has acytochrome c of at least 104 residues, so any given pair of species has more than half itsresidues in common. (Adapted from Creighton, T. E., 1983. Proteins: Structure and Molecular Properties.San Francisco: W. H. Freeman and Co.)HumanChimpanzeeSheepRattlesnakeCarpGarden snailTobacco hornworm mothBaker’s yeast (iso-1)CauliflowerParsnipChimpanzeeSheepRattlesnakeCarpSnailMothYeast Cauliflower0 10 14 18 29 31 44 44 4310 14 18 29 31 44 44 4320 11 24 27 44 46 4626 28 33 47 45 4326 26 44 47 4628 48 51 5044 44 4147 4713FIGURE 5.29 ● This phylogenetic tree depicts the evolutionary relationships amongorganisms as determined by the similarity of their cytochrome c amino acid sequences.The numbers along the branches give the amino acid changes between a species and ahypothetical progenitor. Note that extant species are located only at the tips of branches.Below, the sequence of human cytochrome c is compared with an inferred ancestralsequence represented by the base of the tree. Uncertainties are denoted by questionmarks. (Adapted from Creighton, T. E., 1983. Proteins: Structure and Molecular Properties. San Francisco: W. H. Freeman and Co.)▲5.8 ● Nature of Amino Acid Sequences 145AlaThrAlaAsn GluThrAla? Lys Gly Ala Lys Ile Phe Lys Thr ? Cys Ala Gln Cys His Thr Val Glu Gly ?LysAspGly ? GlyVal Lys Gly Lys Lys Ile Phe Ile Met Lys Cys Ser Gln Cys His Thr Val Glu Gly LysGluAspGly Lys GlyAlaProAncestral cytochrome cHuman cytochrome c1 10 20Val Pro Asn Leu His Gly Leu Phe Gly Arg Lys ? Gly Gln Ala ? Gly Tyr Thr AspGlyLysHis Ser TyrThr Pro Asn Leu His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Thr AlaGlyLysHis Ser Tyr504030Lys Lys Gly ? ? Trp ? Glu Asn Thr Leu Phe Glu Tyr Leu Glu Asn Pro Tyr IleAsnAsnAla Lys LysLys Lys Gly Ile Ile Trp Gly Glu Asp Thr Leu Met Gln Tyr Leu Glu Asn Pro Tyr ProAsnAsnAla Lys Lys60 70Thr Met ? Phe ? Gly Leu Lys Lys ? ? Asp Arg Ala Asp Leu Ile Ala Lys ?LysGlyPro Tyr LeuThr Met Ile Phe Val Gly Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile Ala Lys LysLysGlyPro Tyr Leu80 90 100IleRelated Proteins Share a Common Evolutionary OriginAmino acid sequence analysis reveals that proteins with related functions oftenshow a high degree of sequence similarity. Such findings suggest a commonancestry for these proteins.Oxygen-Binding Heme ProteinsThe oxygen-binding heme protein of muscle, myoglobin, consists of a singlepolypeptide chain of 153 residues. Hemoglobin, the oxygen transport proteinof erythrocytes, is a tetramer composed of two �-chains (141 residues each)and two �-chains (146 residues each). These globin polypeptides—myoglobin,�-globin, and �-globin—share a strong degree of sequence homology (Figure5.30). Human myoglobin and the human �-globin chain show 38 amino acidFIGURE 5.30 ● Inspection of the amino acid sequences of the globin chains of humanhemoglobin and myoglobin reveals a strong degree of homology. The �-globin and �-globin chains share 64 residues of their approximately 140 residues in common.Myoglobin and the �-globin chain have 38 amino acid sequence identities. This homologyis further reflected in these proteins’ tertiary structure. (Irving Geis)146 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureGly Leu Ser Asp Gly Glu Trp Gln Leu Val Leu Asn Val Trp Gly Lys Val Glu Ala Asp Ile Pro Gly His Gly Gln Glu ValVal Leu Ser Pro Ala Asp Lys ThrAsn Val Lys Ala Ala Trp Gly Lys Val Gly Ala His Ala Gly Gln Tyr Gly Ala Glu AlaVal Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp Gly Lys Val Asn Val Asp Glu Val Gly Gly Glu AlaHisArg Phe Lys Gly His Pro Glu Thr Leu Glu Lys Phe Asp Lys Phe Lys His Leu Glu Asp Glu Met Lys Ala Ser GluLeuIleLeu Lys SerArg Phe Leu Ser Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp Leu Gly Ser AlaMetGluLeu Ser HisArg Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp Leu Pro Asp Ala Val Met Gly Asn ProLeuGlyLeu Ser ThrLys His Gly Ala Thr Val Leu Thr Ala Leu Gly Gly Ile Leu Lys Lys Lys Gly Glu Ala Glu Ile Lys Pro Leu AlaLysLeuAsp His HisLys His Gly Lys Lys Val Ala Asp Ala Leu Thr Asn Ala Val Ala His Val Asp Pro Asn Ala Leu Ser Ala Leu SerGlyValGln Asp MetLys His Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp Lys Gly Thr Phe Ala Thr Leu SerAlaValLys Asn LeuHis Thr Lys His Lys Ile Pro Val Lys Tyr Leu Glu Phe Ile Ser Glu Cys Ile Val Leu Gln Ser Lys His Pro GlyAlaSerGln Ile GlnHis His Lys Leu Arg Val Asp Pro Val Asn Phe Lys Leu Leu Ser His Cys Leu Thr Leu Ala Ala His Leu Pro AlaAlaLeuAsp Leu HisHis Asp Lys Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val Leu Ala His His Phe Gly LysCysLeuGlu Val AsnGly Asp Ala Gln Gly Ala Met Asn Lys Ala Leu Glu Leu Phe Arg Lys Asp Met Asn Tyr Lys Glu Leu Gly Phe GlnAlaPheAsp Ala SerThr Ala Val His Ala Ser Leu Asp Lys Phe Leu Ala Ser Val Ser Thr Val Leu Lys Tyr ArgProPheGlu Thr SerThr Pro Val Gln Ala Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Lys Tyr HisProPheGlu Ala HisGlyMyoglobinαβα–chain of horse methemoglobin β–chain of horse methemoglobin Sperm whale myoglobin1 10 2030 40 50 6070 80 90100 110 120130 140 150identities, whereas human �-globin and human �-globin have 64 residues incommon. The relatedness suggests an evolutionary sequence of events in whichchance mutations led to amino acid substitutions and divergence in primarystructure. The ancestral myoglobin gene diverged first, after duplication of aprimordial globin gene had given rise to its progenitor and an ancestral hemo-globin gene (Figure 5.31). Subsequently, the ancestral hemoglobin gene dupli-cated to generate the progenitors of the present-day �-globin and �-globingenes. The ability to bind O2 via a heme prosthetic group is retained by allthree of these polypeptides.Serine ProteasesWhereas the globins provide an example of gene duplication giving rise to aset of proteins in which the biological function has been highly conserved,other sets of proteins united by strong sequence homology show more diver-gent biological functions. Trypsin, chymotrypsin (see Section 5.7), and elas-tase are members of a class of proteolytic enzymes called serine proteasesbecause of the central role played by specific serine residues in their catalyticactivity. Thrombin, an essential enzyme in blood clotting, is also a serine pro-tease. These enzymes show sufficient sequence homology to conclude that theyarose via duplication of a progenitor serine protease gene, even though theirsubstrate preferences are now quite different.Apparently Different Proteins May Share a Common AncestryA more remarkable example of evolutionary relatedness is inferred fromsequence homology between hen egg white lysozyme and human milk �-lactalbumin, proteins of quite different biological activity and origin. Lysozyme(129 residues) and �-lactalbumin (123 residues) are identical at 48 positions.Lysozyme hydrolyzes the polysaccharide wall of bacterial cells, whereas �-lactalbumin regulates milk sugar (lactose) synthesis in the mammary gland.Although both proteins act in reactions involving carbohydrates, their func-tions show little similarity otherwise. Nevertheless, their tertiary structures arestrikingly similar (Figure 5.32). It is conceivable that many proteins are relatedin this way, but time and the course of evolutionary change erased most evi-dence of their common ancestry. In an interesting contrast to this case, theproteins actin and hexokinase share essentially no sequence homology, yet theyhave very similar three-dimensional structures, even though their biologicalroles and physical properties are quite different. Actin forms a filamentouspolymer that is a principal component of the contractile apparatus in muscle;hexokinase is a cytosolic enzyme that catalyzes the first reaction in glucosecatabolism.Mutant ProteinsGiven a large population of individuals, a considerable number of sequencevariants can be found for a protein. These variants are a consequence of muta-tions in a gene (base substitutions in DNA) that have arisen naturally withinthe population. Gene mutations lead to mutant forms of the protein in whichthe amino acid sequence is altered at one or more positions. Many of thesemutant forms are “neutral” in that the functional properties of the protein areunaffected by the amino acid substitution. Others may be nonfunctional (ifloss of function is not lethal to the individual), and still others may display arange of aberrations between these two extremes. The severity of the effectson function depends on the nature of the amino acid substitution and its rolein the protein. These conclusions are exemplified by the more than 300 humanFIGURE 5.31 ● This evolutionary tree isinferred from the homology between theamino acid sequences of the �-globin, � -glo-bin, and myoglobin chains. Duplication of anancestral globin gene allowed the divergence ofthe myoglobin and ancestral hemoglobingenes. Another gene duplication event subse-quently gave rise to ancestral � and � forms, asindicated. Gene duplication is an importantevolutionary force in creating diversity.5.8 ● Nature of Amino Acid Sequences 147Myoglobin β αAncestralβ-globinAncestralα-globinAncestralhemoglobinAncestral globinNC123C129N� -LactalbuminLysozymeHen egg white lysozyme� -lactalbuminHuman milk148 Chapter 5 ● Proteins: Their Biological Functions and Primary StructureTable 5.9Some Pathological Sequence Variants of Human HemoglobinAbnormal Hemoglobin* Normal Residue and Position SubstitutionAlpha chainTorino Phenylalanine 43 ValineMBoston Histidine 58 TyrosineChesapeake Arginine 92 LeucineGGeorgia Proline 95 LeucineTarrant Aspartate 126 AsparagineSuresnes Arginine 141 HistidineBeta chainS Glutamate 6 ValineRiverdale–Bronx Glycine 24 ArginineGenova Leucine 28 ProlineZurich Histidine 63 ArginineMMilwaukee Valine 67 GlutamateMHyde Park Histidine 92 TyrosineYoshizuka Asparagine 108 AspartateHiroshima Histidine 146 Aspartate*Hemoglobin variants are often given the geographical name of their origin.Adapted from Dickerson, R. E., and Geis, I., 1983. Hemoglobin: Structure, Function, Evolution and Pathology.Menlo Park, CA: Benjamin-Cummings Publishing Co.FIGURE 5.32 ● The tertiary structures of hen egg white lysozyme and human �-lactal-bumin are very similar. (Adapted from Acharya, K. R., et al., 1990. Journal of Protein Chemistry 9:549–563;and Acharya, K. R., et al., 1991. Journal of Molecular Biology 221:571–581.hemoglobin variants that have been discovered to date. Some of these are listedin Table 5.9.A variety of effects on the hemoglobin molecule are seen in these mutants,including alterations in oxygen affinity, heme affinity, stability, solubility, andsubunit interactions between the �-globin and �-globin polypeptide chains.Some variants show no apparent changes, whereas others, such as HbS, sickle-cell hemoglobin (see Chapter 15), result in serious illness. This diversity ofresponse indicates that some amino acid changes are relatively unimportant,whereas others drastically alter one or more functions of a protein.5.9 ● Synthesis of Polypeptides inthe LaboratoryChemical synthesis of peptides and polypeptides of defined sequence can becarried out in the laboratory. Formation of peptide bonds linking amino acidstogether is not a chemically complex process, but making a specific peptidecan be challenging because various functional groups present on side chainsof amino acids may also react under the conditions used to form peptide bonds.Furthermore, if correct sequences are to be synthesized, the �-COOH groupof residue x must be linked to the �-NH2 group of neighboring residue y in away that prevents reaction of the amino group of x with the carboxyl group ofy. Ingenious synthetic strategies are required to circumvent these technicalproblems. In essence, any functional groups to be excluded from reaction mustbe blocked while the desired coupling reactions proceed. Also, the blockinggroups must be removable later under conditions in which the newly formedpeptide bonds are stable. These limitations mean that addition of each aminoacid requires several steps. Further, all reactions must proceed with high yieldif peptide recoveries are to be acceptable. Peptide formation between aminoand carboxyl groups is not spontaneous under normal conditions (see Chapter4), so one or the other of these groups must be activated to facilitate the reac-tion. Despite these difficulties, biologically active peptides and polypeptideshave been recreated by synthetic organic chemistry. Milestones include the pio-neering synthesis of the nonapeptide posterior pituitary hormones oxytocinand vasopressin by du Vigneaud in 1953, and in later years, the blood pres-sure–regulating hormone bradykinin (9 residues), melanocyte-stimulating hor-mone (24 residues), adrenocorticotropin (39 residues), insulin (21 A-chainand 30 B-chain residues), and ribonuclease A (124 residues).Solid Phase Peptide SynthesisBruce Merrifield and his collaborators found a clever solution to the problemof recovering intermediate products in the course of a synthesis. The carboxyl-terminal residues of synthesized peptide chains were covalently anchored toan insoluble resin particle large enough to be removed from reaction mixturessimply by filtration. After each new residue was added successively at the freeamino-terminus, the elongated product was recovered by filtration and read-ied for the next synthetic step. Because the growing peptide chain was coupledto an insoluble resin bead, the method is called solid phase synthesis. The pro-cedure is detailed in Figure 5.33. This cyclic process has been automated andcomputer controlled so that the reactions take place in a small cup withreagents being pumped in and removed as programmed. The 124-residue-longbovine pancreatic ribonuclease A sequence was synthesized, and the final prod-uct was enzymatically active as an RNase.5.9 ● Synthesis of Polypeptides in the Laboratory 149150CH3CH3CCH3O CODicyclohexyl-carbodiimideActivatedamino acidCH3CH3CIncomingblockedamino acidCH3O CR2NHCHCOOHBlocking groupR1H2NCHCNHCHCR2Acid H3CCCH3CH3 CIncoming blockedamino acid O CR3NHCHCOOHCH3 CAmino-blockedtripeptidyl-resin particleO CR1NHCHC NHCHCN HCHCR2AcidTripeptidyl-resin particleR1H2NCHC NHCHC NHCHCR2R3Isobutylene +OR3O OOOOOO O O OOO OO OOIsobutyleneCH3CH3CH3CH3Amino-blockeddipeptidyl-resin particle+ CNNR3 NHCHCOOO CNNHActivatedamino acidDipeptide-resinparticle+ CNNO CNNHOCNHNHH2N CHCOR2R1CH3CH3CCH3O CR1NHCHCNHCHCR2OOO OCO2Aminoacyl-resin particleODicyclohexylureaCH2CNHNHOCH3 C O COOCH3CH3NHCHCCH3 C OCH3CH3ClCBocClCNNH2N CRHOHC H2N CRHOC CNNH+OO ODicyclohexyl-carbodiimideActivatedamino acidCO2FIGURE 5.33 ● Solid phase synthesis of a peptide. (inset)Tertiary butyloxycarbonyl chloride (tBocCl) is an excellentreagent for blocking amino groups of amino acids duringorganic synthesis. Dicyclohexylcarbodiimide (DCCD) is a pow-erful agent for activating carboxyl groups to condense withamino groups to form peptide bonds. The carboxyl group ofthe first amino acid (the carboxyl-terminal amino acid of thepeptide to be synthesized) is attached to an insoluble resinparticle (the aminoacyl -resin particle). The next amino acid,with its amino group blocked by a tBoc group and its carboxylgroup activated with DCCD, is reacted with the aminoacyl-resin particle to form a peptide linkage, with elimination ofDCCD as dicyclohexylurea. Acid treatment removes the N-ter-minal tBoc blocking group as the gaseous products CO2 andisobutylene, exposing the N-terminus of the dipeptide foranother cycle of amino acid addition. The growing peptidechain is easily recovered after cyclic additions of amino acidssimply by filtering or centrifuging the reaction mixture.PROBLEMS1. The element molybdenum (atomic weight 95.95) constitutes0.08% of the weight of nitrate reductase. If the molecular weightof nitrate reductase is 240,000, what is its likely quaternary struc-ture?2. Amino acid analysis of an oligopeptide seven residues longgaveAsp Leu Lys Met Phe TyrThe following facts were observed:a. Trypsin treatment had no apparent effect.b. The phenylthiohydantoin released by Edman degradation wasc. Brief chymotrypsin treatment yielded several products, includ-ing a dipeptide and a tetrapeptide. The amino acid compositionof the tetrapeptide was Leu, Lys, and Met.d. Cyanogen bromide treatment yielded a dipeptide, a tetrapep-tide, and free Lys.What is the amino acid sequence of this heptapeptide?3. Amino acid analysis of another heptapeptide gaveAsp Glu Leu LysMet Tyr Trp NH4The following facts were observed:a. Trypsin had no effect.b. The phenylthiohydantoin released by Edman degradation wasc. Brief chymotrypsin treatment yielded several products, includ-ing a dipeptide and a tetrapeptide. The amino acid compositionof the tetrapeptide was Glx, Leu, Lys, and Met.d. Cyanogen bromide treatment yielded a tetrapeptide that hada net positive charge at pH 7 and a tripeptide that had a zero netcharge at pH 7.What is the amino acid sequence of this heptapeptide?4. Amino acid analysis of a decapeptide revealed the presenceof the following products:NH4 Asp Glu Tyr ArgMet Pro Lys Ser PheThe following facts were observed:a. Neither carboxypeptidase A or B treatment of the decapep-tide had any effect.b. Trypsin treatment yielded two tetrapeptides and free Lys.c. Clostripain treatment yielded a tetrapeptide and a hexapep-tide.d. Cyanogen bromide treatment yielded an octapeptide and adipeptide of sequence NP (using the one-letter codes).e. Chymotrypsin treatment yielded two tripeptides and atetrapeptide. The N-terminal chymotryptic peptide had a netcharge of �1 at neutral pH and a net charge of �3 at pH 12.H2COCHSNHAiO OHONCCH2COCHSNHAiONCCf. One cycle of Edman degradation gave the PTH derivativeWhat is the amino acid sequence of this decapeptide?5. Analysis of the blood of a catatonic football fan revealed largeconcentrations of a psychotoxic octapeptide. Amino acid analysisof this octapeptide gave the following results:2 Ala 1 Arg 1 Asp 1 Met 2 Tyr 1 Val 1 NH4The following facts were observed:a. Partial acid hydrolysis of the octapeptide yielded a dipeptideof the structureb. Chymotrypsin treatment of the octapeptide yielded
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