PART IV BIOCHEMISTRYAcid-Base Equilibrium and Buffering > Homeostasis requires that the pH of biologic fluids be maintained at a constant and characteristic value. The control of pH in body fluids and subcellular compartments is achieved by a number of biologic buffers. The most important buffers in physiologic systems are bicerbonete, phosphate, and proteins. UME consist of a weak acid. and its conjugate base that are in equilibrium and are,able to donate an s from the surrounding medium, The | telationship between pH, pKa, and the concentration of the conju- gate acid and base of a buffering system is described by the uurbances in the acid-base balance can lead toledo plasma pH of less than 7.35) oak loss (2 plasma pH of greater than 7.45). Ths chapter reviews the concepts underlying acid-base equilibrium, the pH of various body fluids, and the properties of the buffer systems that resist fluctua- tions in pH. ACID-BASE EQUILIBRIUM Proterr Dea ‘The most appropriate biochemical definition for acids and bases is that acids a ‘acceptors. Many important com- pounds found in biologic fluids, including amino acids, lactic acid, and ace- toacetic acid, are weak acids. |. Properties of weak acids. Weak acids, in contrast to strong acids such as HCl or HzS0g, are not completely dissociated under the conditions that exist within biologic systems. The undissociated form (HA) that retains the proton is known as the <@hijugiateladid, whereas the corresponding deprotonated form (A) is a conjugatelbase’that can accept a proton to recreate the acid. The reaction that describes the dissociation of a weak acid is shown in Figure 1-1. KAPLAN MEDICAL 325NBDE 1: BiocHEMIsTRY we e conjugate acid conjugate base Figure 1-1. Dissociation of a weak acid. 8. The Henderson-Hasselbalch equation shown in Figure 1-2 describes the relationship between pH and the concentration of a weak acid and its conjugate base. In this equation pHs defined as the negative log of (18) and BKGls defined as the negative log of the dissociation constant Ka where K, = "7. For any weak acid the py s a constant that is Characteristic of that particular ionizing group. The Henderson- Hasselbalch equation is useful because It provides @ meaningful de tion of pky and it permits calculation of the amounts of the undissociat- ed acid and its conjugated base at any pl. tent (Ad - PH = pK + loa" ay Figure 1-2. Henderson-Hasselbalch equation. Were 1. A funciona definition of pk Ean be deduced from the Headeron- ‘cooly ofthe equation: When the Hasetbleh equation, The oX; of ary ining group canbe defines: @ eee el the a mae. as the pH where the concentrations of the acid (HA) and its conju- Ke open earpepi ee gate base (A) are equal. When the pH is equal othe pKa ofthe nant. acid, the concentrations of HA and A” are equal. Inspection of the ‘equation indicates that when [HA] = [A], the last term of the equa- ton becomes zero and the equation reduces to pH = pk. 2. The logarithmic nature of the Henderson-Hasselbalch equation indi- cates that small changes in pH will bring about large changes in the relative concentrations of HA and A. These effects are summarized in Table 1-1, where, is considered an ‘example of a weak acid. The same principles apply for any weak acid, leva Unit results in 2 10-fold change in the ratio of ‘onlugate acid to conjugate base Table 1-1. Effect of pH on the relative amounts of lactic acid and lactate, pH Ulactic acid}/tlactate-] 28 100 38 10 pe anes a ae OW eT] 38 a1 68 : 0.01 326 KAPLAN MEDICALAciD-BAse EQUILIBRIUM AND BUFFERING TITRATION AND BUFFERING PROPERTIES OF WEAK ACIDS {TtfBHI can be defined as the addition of a strong base,to.a.weak.acid (or ‘a strong acid to a weak base) for the purpose of buffering the solution, i.e., maintaining a constant pH. |. Titration curve. The adltion of a strong base such as sodium hydroxide t0.a weak acid may be written as: HA + NaQqH =———> NaA + H20 ‘As NaOH is added, the protons released by the weak acid combine with hydroxide to form water. The continued addition of sodium hydroxide progressively decreases the [H*] and increases the pH. A graph of the titration of a weak acid with a strong base is shown in Figure 1-3. hes Butloring range pKa HA o 05 4.0 NaOH (equivalents) Figure 1-3, Thration curve for a weak acid. [At the midpoint in the titration, half of the acid has been neutralized and therefore, [HA] = [Ar]. The Henderson-Hasselbalch equation shows that when [HA] = [4°], the pH of the solution is equal to the pKa of the weak acid 8. Buffering capacity. Buffers are mixtures of weak acids and their conju- gate bases. The capacityof alberto resist chang In Hs dependent ‘on two factors: the 1d the pH at which itis Used. A butler is most effective when its used in a pH range near the Ph. ts clear from Figure 1-3 that in the pH range defined by 1.0 pH unit above and below the pk, the addition of either strong acid or strong base will result in minimal change in the pH. KAPLAN MEDICAL. Nore The best buffering occurs when pi 'pH, resulting in 50% of acid and 50% of ‘base, This system can then handle either {an addition of base or an adtition of acid, 327NBDE 1: BiocHEemistRY BRIDGE TO PHysioLoGy (EIBBIR is frequently found in the cat- aipic site of enzymes where the imida- ze ring can donate or accept protons in the formation and breaking of bonds. BRIDGE TO PuYsioLoGy RGB8I8 2 piasma p ‘metabolic a or from ioniehart results in the, 1, (respiratory. Saroov ame 245, cen result from a loss ue to excessive afc alkalosis) or from (byoervemiation (espiretryakalst) 328 PHYSIOLOGICALLY IMPORTANT BUFFERING SYSTEMS EEERRIANT BUFFERING SYSTEMS The three most importantbutfering syste in biologic systems are proteins, bicarbonate, and phosphate. ‘The conjugate acid-base pairs and the pka values for these buffers are summarized in Table 1-2. The isj6FlBUFfEe in plasma and interstitial fluid is bicarbonate, whereas protein and organic phosphate esters are the major butfers of intracellular fluid. ‘A: Protein buffering systems. The @/t686I/6FIGll8 contains high concentra- tions of proteins with amino acid side chains that are weak acids and bases. These side chains impart great buffering capacity to proteins. The «seidieamRinoraeid (glutamic and aspartic acids) and the basicaminovacis. (lysine, histidine,.and_arginine) all contain ionizable side chains (dis- cussed in the next chapter). However, histidine fs the only amino.acid with capacity at physiologic pH. ‘The imidazole side chain of histidine has a pkg that ranges from 5.6-7.0, depending on its microenvironment within the protein. Table 1-2, Properties of major physiologic butters. Buffering system PK, Protein systems: Histidine side chains 5.67.0 Bicarbonate system: HCO371CO2, 61 Phosphate systems: HPO4?/H2PO4- 68 Organic phosphate esters 65-75 8. Bicarbonate buffering system. The bicarbonate-CO2 system is the most important fluid at its normal value of 7.4, of igure 1-4 summarizes the properties of ‘this buffering system. Gartimieeid (12C03) i the proton donor, Blears, ‘BonatelanioniXH1CO;>) is the proton.acceptor, and the pK, for this reac- tion is 6.1. The strength of this buffering system lies in the ability of car- bonic acid to be converted to carbon dioxide. H2CO3 SHY + HCO pk, HY + HCO = HCO) ed COP + H30 Figure 1-4. The bicarbonate/CO> buffering system. . Phosphate buffering system. Intracellular fluids contain high concentra tions of inorganic phosphate and many organic phosphate esters that contribute significantly to the buffering power of the cytosol. The phos- hate buffering system is of litle importance in plasma and interstitial KAPLAN MEDICAL zBAcip-BASE EQUILIBRIUM AND BUFFERING @ tid because of the low concentrations of phosphates in extracellular fluids. The phosphate buffering system consists of HPO,” as the pro- ton donor and HPO3* as the proton acceptor. The pKa of 6.8 is suffi- ciently close to the normal intracellular pH to make it an ideal buffer in those fluids that contain high concentrations of phosphates, such as red_ blood cells and kidney tubules. KAPLAN MEDICAL 329Proteins, Enzymes, and Coenzymes Proteins are linear polymers. of aming acids, each having distinct chemical and structural properties. They are the largest class of molecules found in living systems and are responsible for most of the diverse functions of the cell. They act as transport proteins, storage proteins, hormone receptors, structural proteins, regulatory proteins, and enzymes. Each protein has a unique amino acid sequence that determines its precise three-dimensional conforma- tion and specifies its function. The most versatile class of the pro- ‘teins are the enaymes, which act as biologic catalysts, enhancing the rate of the chemical reactions occurring in cells. Most enzymes catalyze only one reaction, and their catalytic activities may be stringently regulated, allowing the cell to control and coordinate its metabolic pathways. The function of many enzymes reqi sa cofactor—a metal or a small organic coenzyme derived from a vita- min precursor. This chapter will review the structure and properties of amino acids that are found in proteins, the kinetic and regulato- ry properties of enzymes, and the relationship between coenzymes and their vitamin precursors. AMINO ACIDS: THE BUILDING BLOCKS OF PROTEINS ‘A. Amino acid structure. The building blocks for proteins are the 20 com- mon amino acids that are encoded in the DNA of the cell. 1. General structure. Nineteen of the twenty common amino acids can be represented by the structure shown in Figure 2-1. They all have a an amino.group, ‘and a hydrogen atom, The amino acids differ from one another only in the chemical nature of the side chain (R). The only amino acid that KAPLAN MEDICAL 331NBDE 1: BiocHemistay Iw a Nurswete Hydrophilic amino acid side chains Positive Negative ‘Neutral bys Asp Ser Arg Glu Thr is Os Met Asn Gin In a Nursnece #55 bridges: Qstine * Collagen triple heli: Hydraxyprotine * Signal transduction: Phosphotyrosine Phosphoserine Phosphothreonine 332 ismot described by this structure fpreling, an imine acid in whch the @) side chain forms a cyclic structure with the amino group. eS 1 R —Ca—COOH 4 Figure 2-1. General structure of a-amina acids. 2. Classification. The amino acids can be classified as either hydropho- bie or hydrphill, depending on the care with which th chain interact with water Ths parar method of estention espacial nel whan considering amine acs athe bulding backs SR eaiae erate erie wid his doe aiaeelg the protein ols Into more compact stucures In general qroteing fold sa. tnat tha hydcoahoblc se chains ae In the Intise arate nocaeyiee ae and the epic seoarenceteoee > Hycrophole ere ads hve side chins with aohatic groups or aromatic ring structures. . Hydrophilic amino acids have side chains that contain On.Ny or $ atoms. Some of the hydrophilic side chains are charged at physio- logic.pl. The acidic amino acids (aspartic. and glutamic acids) have carboxyl groups that are negatively charged, while the basic amino acids ( ine, a ine) have nitrogen atoms that are positively charged. Other hydrophilic amino acids with uncharged side chains contain hydroxyl groups (serine and threo- nine), a sulfur atom (qsteine), or amide groups (asparagine and glutamine 3, Modified amino acids. In addition to the 20 commen amino acids that have thelr origin in the genetic code, proteins often contain ‘ther amino acids that are generated by modification of the common amino acids after the protein has been synthesized. This process is «called post-translatis fication. 2. Cystine is formed in proteins by the reaction of two cysteine side chains to form.a.disulfide linkage. tis found most frequently in lular proteins where the disulfide bond stabilizes the three-dimensional structure of the protein. This s the only cove- lent cross-linkage found in proteins. b. Hydroxyproline is formed in an oxygen-deper reaction that occurs in fibroblasts. Itis found in collagen, where it stabilizes the triple helical structure. KAPLAN MEDICALB. Properties of amino Proteins, ENZYMES, AND COENZYMES & Phosphotyrosine, phosphoserine, and phosphothreonine are formed by transferring phosphate from. ATP.t0,the.hysroyy! stup of serine, tyrosine, or threonine. These amino acids are found in many enzymes and protelns, where they serve as regul- tory signals, ‘The properties luence he properties of proteins are influenced by the properties of their constituent amino acids. In particular, the stereo: wa d,ionic properties of amino acids have an impact on the icture andfor properties of the protein. 1. Stereochemistry. The a-carbon atom of all amino acids except glycine is linked to four different chemical groups, making the a-carbon ‘atom an asymmetric center. As shown in Figure 22, an asymmetric center has two stereoisomers (enantiomers) that are mirror.images of er and ar an bs ids. Only L-amino acids are incorporated into proteins. coo” cyto CH, L-alanine Figure 2-2. stereoisomers of an a-amino acid, 2, tonic properties. Depending on the pH, an emino acid may.have no, _net charge, or it may have either a positive or a negative charge, The ‘pH at which @ molecule is electrically neutral is defined as the isoelee tricpoint (01). The equilibrium between the ionic forms of an amino ‘acid is shown in Figure 23. The pK, values for the «-carboxyl and o- ‘amino groups are approximately 2.1 and 9.8, respectively. At a pH below the pK, the protonated species predominates, whereas at a pH above the pk,, the deprotonated species predominates. The pH for the amino acid shown in Figure 2-3 is the average of the two PK values. At neutral pH, the species that has both positive charge and negative charge is called azzwitterion; KAPLAN MEDICAL Nore There are other amino acio ike RHINE and éitrilling that play important roles in the cell but that are not part of the build- ing block of 20. 333NBDE 1: BiOcHEMIsTRY In A NutsHete 1? structure: » Amino acid sequence P structures» ahelix * Bsheer 3° structure: + Overall protein structure + How achelic and Besheet fold with respect to each other 4° structure: * Mutiple polypeptide chains ‘+ How chains fold with respect 10 each other 334 NI *NI NH, ies meer pkg = 98 e R—CH—COOH 81 GH—c09 ———* R—CH—CO0- ‘Net charge Net charge = +1 PH=1 Figure 2-3. The ionic equilibrium for an a-amino acid. The dliie™SRlBBSIESFRING acids also have ionizing groups in thelr side chains. aspanticlandiglutamielaelds have side chain carboxyl gr0Ups with a pKa of approximately 4.0, and are negatively charged at physiologic pH. stead arGininghave side chains with protonat- ed nitrogen atoms having pky values of approximately 10 and 12 ‘fespectively, and are positively charged at physiologic pH. Aistidinig hhas an imidazole side chain with a pKa of approximately 6.0, a value sutficiently close to the physiologic pH to allow some of the histidine side chains to be positively charged while others have no charge. The ratio of positively charged histidine side chains to uncharged side chains at any pH can be calculated from the Henderson-Hasselbalch {equation (described in the previous chapter). PROTEINS Each protein has a unique three-dimensional structure dictated by its amino ‘acid sequence and responsible for the highly specific function of the protein. A. Structural features. The amino acids in a protein are linked together by ‘peptide bonds in which the a-carboxyl of one amino acid is linked to the ‘a-amino group of another amino acid (Figure 2-4). The peptide (amide) bond has partial. double-bond. character, making it planar and rigid, ‘thereby imposing limitations on higher orders of structural organization ina protein. 1. Primary structure. The sequence in which the amino. acids occur in the polypeptide is defined as the primary structure. This sequenc ‘encoded by the DNA, and it determines how the protein folds into a ‘more compact three-dimensional structure. H[o R ° + Iii i il H,N—C 40 —NH4CH 4c —NH -LcH —C —coo” 1 il | R 0 R Figure 2-4. The peptide band. KAPLAN MEDICALPROTEINS, ENZYMES, AND COENZYMES. 2, Native conformation. The final structure that a particular protein assumes proceeds through several levels of organization. In contrast to the primary structure, which was stabilized by strong covalent bonds, higher orders of structure are stabilized by numerous weak noncovalent interactions. ‘8. Secondary structure in proteins is organized around the polypep- tide backbone and is stabilized by large numbers of hydrogen bonds formed between the amide hydrogen atemef one psmtide bond and the carbonyl oxygen atom of another. Proteins contain ‘two major types of secondary structure: achelicalistructure, which is stabilized by intrathain hydrogen bonds, and Basheetistructure/) Which is stabilized by interchain hydrogen bonds. In some fibrous proteins, the highest order of structure is secondary. These pro- teins are elongated asymmetrical proteins that frequently function a structural components of cel and tisues. simple combinations cof afew secondary structure elements are called qui An exam- ple of a motif would be alfiaieBimotifithat can be used to connect f Js. The achelix in the middle of the two B aceon iealaetege. b. Tertiary and quaternary structure. Segments of secondary struc ture in globular proteins associate with one another and fold e into a tertiary structure that i stabil tos between amin acts shi. The fundoneta int of | tertiary structure Is the domain, a part of the polypeptide chain that folds into a stable tertiary structure, Proteins that are com posed of more than one. polypeptide.chain have quaterney structure. Both tertiary and quaternary structures are stabilize by ionic and hydrophobic interactions and by hydrogen ate between side chains. Bt Oeeetion dined asa on chit cesta iesrg iat capa a ERR ey of Se Pee att peeromtion raj be Casey at eareae ta i eres ect neste nwa res clu al Many of the proteins in skin, bone, muscle, and hair perform structural Bes re eee ce aaa cag Gn eer eine malielaaemtet sor oe eetosn: EG Sourcreegrera pre Se ee forrpeat el noel res deriva Ulatory roles in the cell; and enzymes function as biologic catalysts. ENZYMES e Virtually all biologically important reactions are catalyzed by enzymes. Most of these reactions occur under very mild conditions of temperature and pH KAPLAN MEDICAL 335NBDE 1: BlocHemistay that are compatible with living organisms. In the absence of enzymes, reactions in the cell would proceed at insignificant or undetectable rates. Enzymes differ from inorganic catalysts in thelr specificity, catalytic eff- ciency, and regulatory properties, ‘A. Classification. Enzymes are divided into six different classes on the | basis of the types of reactions they catalyze (Table 2-1). All of the thousands of different reactions occurring in cells fall into one of these six types of reactions. Table 2-1. The six classes of enzymes. “Class ‘Type of Reaction Oxidoreductases Oxidation-reduction reactions Frequently use coenzymes NAD+, FAD, NADP, or 02 as electron acceptors Rehudmasnase. guidase, ceductase) Transferases Transfer of @ chemical group from a donor to an acceptor Groups transferred include amino, carboxyl, acyl, glycosyl, phosphoryl CTransaminase, kinase) Hydrolases Cleavage of a bond between carbon and some other atom by the addition of water (Prsteaas, Puosphatare. amass) lyases Nonhydroiytic cleavage of carbon-carbon, carbon-sulfur, and some carbon-nitrogen bonds (Aldolase, decarboxylase, dehydratase) Isomerases Interconversion of isomers pimerase, mutase) Ligases Formation of bonds between carbon and oxygen, nitrogen, or sulfur atoms in reactions that require energy (Carboxylase, thiokinase) 336 8. Specificity. The high-level specificity of enzyme-catalyzed reactions occurs because enzymes have active sites composed of a small number of amino acid side chains. The side chains come together to form a three-dimensional site fon the surface of the enzyme that is complementary to the structure of the substrate. Some of the amino acid side chains in the active site participate in binding the substrate to the enzyme; others act as catalytic groups and enhance the rate of the reaction by acting as acids, bases, or nucleophil. KAPLAN MEDIC:© Catalytic properties. The eneray profile of a chemical reaction, as it pro- ceeds from substrate to product, is shown in Figure 2-5. The highest point on the curve isthe energy of the ffaisitianistate an intermediate whose properties resemble bi luc. The, barrier created by the transition state is also known as the aetivationy ‘energy|(AG") of the reaction. To initiate the reaction, energy must be expended to overcome the energy barrier. The rate of a reaction is ‘inversely proportional te. the maanituds ofthe activation energy. As shown by the dotted line in Figure 2-5, enzymes increase the rate of 2 reaction by decreasing the activation energy barrier. They have no effect ‘on the equilibrium constant for the reaction, which is related to AG, the ‘energy difference between the products and substrates. Transition state pe minus enzyme plus enzyme. Free energy Reaction progress. Figure 2-5. Energy profile fora catalyzed and uncatalyzed reaction. D. Kinetic properties of enzymes. (KIRIG isthe study of the rate at which, chemical reactions occur 1, Factors affecting the rate of enzyme-catalyzed reactions include tem- ncentration, and substrate concentration. Typical rate responses to these factors are shown in Figure 26. 2a. Temperature. The rate of most resctions increases approximately ‘wo-fold.with.10:C.inciease.jo.temperature. For enzyme-cat- alyzed reactions, however, there is an optimum temperature beyond which the rate rapidly decreases due to denaturation of the b. pil The optimal activity of most enzymes occurs between, pH.S and 9. The shape of the rate-vs reflects different ioniza- tion states for specific amino acid side chains that are required for substrate binding of for catays Enzyme concentration. The rate of an enzyme-catalyzed reaction directly proportional to the concentration of enzyme, provided KAPLAN MEDICAL PROTEINS, ENZYMES, AND COENZYMES 337NBDE 1: BIOCHEMISTRY ‘STEP up the reaction rate: Substrate concentration Temperature Enzyme concentration PH In a NursHece Vmax Koy: 338, Theoretical rate when all enzymes are working Substrate concentration at which the reaction is running at half of the maximum rate (where V= 1/2 Vinay) the substrate is present (ficient to saturate the binding sites. A 5 oN Tae mi c = 5 Bs @ Frist order =o Suberae Ce ee Figure 2-6. Factors affecting the rate of enzyme-catalyzed reactions, 4. Substrate concentration. At very low concentrations of substrate, first-order kinetics are observed, withthe rate being directly pro- portional to [S]. When the concentration of substrate is suffi- cently high that all of the binding sites are occupied, zero-order kinetics are seen, with the rate being independent of (SI. The Michaelis Menten equation is an expression thet quantifies the relationship between the rate of an enzyme-catalyzed reaction and ‘the substrate concentration. In deriving an equation for the curve shown in Figure 2-6 (0), it is assumed that the limiting rate observed at an infinitely high substrate concentration is.due.to.a finkte.number df sites on the enzyme, and that when all of the sites are occupied, ‘the maximum rate is obtained. The Michaelis-Menten expression for velocity is illustrated by the following equation: Vmax} Km +[5] In this expression, Vm and Km are constants that ae characteristic of the enzyme. They ae defined 2 follows: a. The iMiaximumvlOKity, Vmax, is the rate obtained when all of the enzyme is present as an ES comples, with substrate bound tothe active site. The Vig increases a5 the concentration of enzyme increases. . The Km sthe\substratesconcentration that is required to achieve half of the maximum velocity. These relationships are shown in Figure 2-7. Rough estimates of the Vinay and Ky values can be KAPLAN MEDIKAPLAN MEDICAL PROTEINS, ENZYMES, AND COENZYMES obtained from this figure. However, values for both Vinax and Km ‘obtained from this type of graph are inherently subject to large terrors due to the shape of the curve; the value for Vmax must be extrapolated from an infinitely large value for [S]. Much more accurate values of these two kinetic parameters can be obtained from secondary plots as described below. Kp, is independent of ‘the enzyme concentration. (s1—> Figure 2-7. Dependence of rate on substrate concentration. 3, Lineweaver-Burk plot. A more useful graph for obtaining values of Km and Vmax can be obtained by taking the reciprocal of the Michaelis-Menten equation and rearranging it to give the Lineweaver- Burkequation: Dee a V™ Vinax "5 * Vmax ‘As shown in Figure 2-8, this equation describes a straight line when Wis plotted against 1S]. The slope of the line is equal to Km/Vmax, the intercept of the line on the y-axis is equal to Vmax, and the Intercept on the x-axis is equal to ~1/Kmy. Note Remember that the equation for a straight line is y = mx + b, where m= slope and b = y intercept 339NBDE 1: BlocHemistay Iw 4 Nurswett Lineweaver-Burk plot seas sy ras N= trate slope KoNVinae intercept: =H spitercept Wax og a ce 's) Figure 2-8. Lineweaver Burk pot 4. Significance of Km and Vmax: Each of these kinetic constants reflects an intrinsic property of the enzyme. 2s related to the afin the.enzmefocthe ubstete, The gy Wate lower the.value for Kmy the higher the affinity of the enzyme for AE ee the substrate, and vice versa. Frequently, the Km value of an sen AO, | fenzyme for its substrate is approximately equal to the substrate # Kn > T substrate affinity concentration found.in the cell. © TnL substrate affinity b. Vmax is related to the efficiency with which substrate.is converted to product when all of the active sites are occupied. Some | ‘enzymes, such as catalase, are highly efficient, with the rate of product formation approximating the rate of substrate binding; ‘others, ike RNA polymerase, are much less efficient at converting substrate to product. E, Inhibitors of enzyme activity. Much of toxicology and pharmacology is based on the principles of enzyme inhibition. Many metabolic poisons, such as insecticides and nerve gases, are enzyme inhibitors. Similarly, ‘many drugs are inhibitors of key enzymes in metabolic pathways, and often have structures similar to the normal enzyme substrate. There are two large clases of enzyme inhibitors: reversible and irreversible. 1. Reversible inhibitors alter the kinetic properties of an enzyme by binding noncovalently Pease TITTY jh. multiple eee with amino acid side chains. The effect of a reversible inhibitor is removed by its dissociation from the enzyme. There are three types of reversible inhibitors: competitive, noncompetitive, and uncompetitive. 340 KAPLAN MEDICALPROTEINS, ENZYMES, AND COENZYMES 4 Competitive inhibitors are structural analogs ofthe substrate and compete with the ive site. The ctfect of a competitive inhibitor can be overcome by increasing the concentration of substrate. The kk for the substrate is increased (ie, since both inhibitor and substrate are competing for the same site, more substrate is needed to reach half maxi- tna vlc) ag rem sncange.Comeatie ston are. -weaver-Burk plots (Figure 2-9), in Which the slope js increased but the intercept on the 1/V axis is unchanged. The intercept on the.acaxis.is shifted. closer to zero (corresponding to an increase in Kr). plus inhibitor ‘minus inhibitor 0 4 s Figure 2-9. Lineweaver-Burk plot of competitive inhibition. 'b, Noncompetitive inhibitors bind to some site other than the active site and are not structural analogs of the substrate. ‘The MiRGKlof the reaction is decreased, but them for the substrate remains unchanged. The effect of a noncompetitive inhibitor cannot be overcome by increasing the substrate concentration. In a Lineweaver-Burk plot Figure 2-10), the intercept on the 1/V axis is increased (corresponding to a decrease in Vmax), and the inter- cept on the 1/5] axis is unchanged © Uncompetitive inhibitors bind directly to the enzyme, substrate complex, but not to free enzyme, This bind causes @ conforma: tional change at the active site that renders the enzyme inactive. Both MimaylamdiKy, are changed. Both Nggylandiki,, decrease, but the siope of the inhibited reaction is parallel with the slope of the Uninhibited reaction (K,/Vimax)- Lineweaver-Burk plots at various concentrations of the inhibitor will be a series of parallel lines. KAPLAN MEDICAL In a NursHece ‘Competitive inhibition: © Kn Vmax unchanged Slope increases eintercept is unchanged -cintercept shifts to the right In a Nurswece ‘Noncompetitive inhibition: Km is unchanged Wr Slope increases intercept shits upward 2cintercept is unchanged Iw a NursHece Uncompetitive inhibition: + Kmand Vmarare changed *© Slope remains the same ++ ‘intercept shifts to the eft = jrinterceot shifts upward 341NBDE 1: BiocHemistrY Cuimicat Conreare eRe i caused by leads nhbtony effect on a number of eneymeshovieg esentiasulycy groups intel acthe sites. The suiffydry! groups react cova- ieniy with lead, resulting in treversble inactivation ofthe enzyme. Two gprmesin the pathy of eet E ‘are inactivat els of lea, resulting in decreased synthe- sis of heme and hemoglobin and increased excretion of aminolevulnic ‘acid, Treatment with chelators such as penicillamine decreases the toxicity by forming nontoxic complexes with lead. In 4 NursHete Allosteric regulation: + Actvotors: 4 KFT Vow + Inhibitors: KFS Vg 342 plus inhibitor minus inhibitor 0 4 1s} Figure 2-10. Lineweaver-Burk plot of noncompetitive inhibition, 2, Irreversible inhibitors covalently bind to.the enzyme, resulting in per- ‘manent inactivation of the enzyme. Some irreversible inhibitors are referred to as “1 8" oF "sul ates” because they bind to the enzyme in ning to uni is; however, the catalytic cycle is never com- pleted because they become covalently linked to the enzyme. The effect of irreversible inhibitors on the kinetic parameters of an ‘enzyme is identical to that ofa noncompetitive inhibitor. F. Regulation of enzyme activity, Homeostasis requires that the rate of metabolic pathways be carefi dinated with one another. This is typically achieved by the regulation of one or two key enzymes in each pathway. The key enzymes that are regulated usually «catalyze either the rateclimiting reaction in the pathway or a reaction that is essentially irreversible. There are four major mechanisms for con- trolling the mes. 1. Allosteric regulation. The activity of allosteric enzymes is regulated by the reversible bin of an effector molecule to a site other than ‘the active site. Substrate saturation curves for allosteric enzymes are usually sigmoidal (Figure 2-11). Allosteric effectors can be either pos- ive (activators) or negative (inhibitors) and act by. altering either the Ky Vmae OF BOtH, AGtivators either decrease the Kn oF increase the Vmaxs While inhibitors increase the Km or decrease the Vmax ‘Common types of effectors include end product of pathways Or mol. ‘ecules that reflect the energy state of the cell (ATP, ADP, AMP, NADH, NAD*, acetyl-CoA), KAPLAN MEDICALPROTEINS, ENZYMES, AND COENZYMES SI Figure 2-1. Substrate saturation curve for an allosteric enzyme. 2. Covalent modification. The activity of many enzymes is regulated by phosphorylation/dephosphorylation cycle in which a specific serine, threonine, or tyrosine side chain becomes modified (Figure 2-12). ‘The addition and remoyal.of the phosphate group are catalyzed by a family of protein kinases and protein phosphatases that respond to hormonal stimulation of cells. Phosphorylation can increase or decrease the activity of an enzyme, or it properties: ——SOsC*=~CS 3. Isoenzymes. Isoenzymes are different proteins that catalyze the seme reaction, but have different catalytic and regulatory properties and frequently differ in tissue and/or organelle specific. The appearance of tissue-specific isoenzymes in plasma is of diagnostic value in identi- ‘ying sites of tissue damage. Protein ate Kinase er oe me fn Phosphatase Figure 2-12. Regulation of enzyme activity by covalent modification KAPLAN MEDICAL fer the regulatory Cuimicat ConreLare GreAHT ACERS (.e,, hormones) activate receptors via the nevay an (as coma ignaticionn goth ‘many cancers, this pathway is often dis- regarded and the growth signal is always (on, causing unregulated cel proliferation. 343NBDE 1: BiocHemistRY 4 Induction and repression of enzyme synthesis. since enzyme activity @ i vec proportional tothe amount of enzyme present, one way to regulate enzyme-catalyzed reactions isto alter the rte at which enzymes are synthesized This type of regulation is frequently med Sec eee ae crienes Oot at nthe slo increase or decrease the rate of transcription, and secondary, po- tein synthesis. (COENZYMES AND VITAMINS Most enzymes that catalyze transfer reactions or oxidation/reduction reac- tions require a coenzyme that serves as an intermediate cartier of some spe- cific functional. group. @oenzymes are small organic. molecules that, are more stable than proteins. Coenzymes are derived from vitamins. (Vitamin) cannot be synthesized de novo by human tissues and must therefore be sup- plied by the diet. In addition to dietary sources, some vitamins are also syn- thesized by bacteria normally present in the intestinal flora. Vitamins may be classified as either water soluble or fat soluble. All of the water-soluble vitamins and some of the fatsoluble vitamins serve as precursors for coen- zymes. Other fat-soluble vitamins control the rate of transcription of spec ie genes, thereby influencing the rate of enzyme-catalyzed reactions by altering the amount enzyme present in the cell [A Water-soluble vitamins e Cuimrcat Correvare ee 1. Niacin, also known as yitamin 83 or nicotinic acid, is present in whole use ‘include tior in grains, meat, and nyt producing the clinical triad o ricotinamide, which is then incorporated into the coenzymes AID Ds: diarrhea, dermatitis, gis and deren. and WABB#! These coenzymes are very. important in both lipid and uckeaiet TD eee carbohydrate metabolism, where they act as carers of hydride ions (ewo electrons and @ proton) in oxidation and reduction reactions. NAD¢ is generally involved in oxidative, catabolic pathways and is more concentrated in mitochondria than in cytosol; NADPH is used in reductive, anabolic pathways and is found primarily in the cytosol 3 2. Riboflavin, vitamin 82, is present in organ meat, whole grains, and — dairy products. The two coenzymes derived from ribofiavin are MN | and FAD, both of which act as carriers of hydrogen atoms in oxidation | and re reduction reactions. These coenzymes are important in the oxida- | tion of carbohydrates, lipids, and amino acids, and are found primarily | in the mitochondria. Human riboflavin requirements increase with increased protein utilization during growth periods, pregnancy, lacta- tion, and wound healing. Patients with a Gsfigiehe) of riboflavin develop lesions of the lips; mouth-skin, and genital 3. Thiamine, vitamin 8, is present in meat, beans, peas, and grains. The coenzyme derived from thiamine is thlamine: pyrophosphate, which 348 KAPLAN MEDICI|. Pyridoxine, vitamin Bs, ire Fasano tll gt ata ghoul enzymes requiring thiaminelpyrophosphate are pyruvate dehydroge- te Lea arc Tae dehydrogenase, and transketolase. eae present in many foods, including most fas epee Tee See SI ote Nain pyridoxaliphosphate) which acts as a carrier of amino groups in ae Scene: areas yee regres fain Orv prloroed exponen eae tig ot baton akc comes emre atoees See ise ee ds marten ae nea ease ees ci aT ae Seles te coonines seried sal he Oa aaa Mer a 6 Geshe of sol igs Se pace eee TSS ee eae nent ct ge a aes egy views Ca ee eee bore earner te Gt acieer iitey ak wera megane mies ta ses Hilpeais Wintel beawia: reo eer one ee STS Ae habe bs connie linked a apm geete enzymes. (BiOtin acts as 2 . " groups for three k lyze carboxylation reactions. The ‘enizymes and the pathways they participate in are: te car lase (gluconeogenesis), acetyl-CoA carbonylese, (fatty acid synthesis), and propionyl-CoA carboxylase (branched chain amino acid catabo- lism). Biotin deficiency is rare. Excessive consumption of raw egg sorption due to the presence of a biotin-binding prot “Antibiotics that alter the intestinal fora can also lead to biotin deficiency. Symptoms of Include alopecia, skin and bowel inflammation, and muscle pain. . Folic acid is present in liver, fresh fruit, and leafy green vegetables. ‘The coenzyme form of this vitamin is tetrahydrofolie acid (TF), which acts as a carter of 4 jetabolism, TF ca one-carbon units (except carboxyl groups) at all stages of oxidation. The coenzyme is imy in. amino acid lism and in the syn- thesis of purines. es. The donors of the one-carbon nits are serine, histidine, glycine, and tryptophan; the acceptors are intermediates in the pathways of purine and pyrimidine nucleotide synthesis. Vitamin C, or ascorbic acid, is found in fresh fruits and vegetables This vitamin exists in both oxidized and reduced forms, with the reduced form being the active form. Vitamin ¢ facilitates iron absorp- ‘tion from the 1Iso required in a number of hydroxylation KAPLAN MEDICAL Proteins, ENZYMES, AND COENZYMES. CuINtcaL ConReLaTe ead to periph- 7 (dry beriber). More severe vitamin depletion leads to bight utput card faire (wet berber and the demerit, end ophithalmane- a ofthe WernickeKorsakotscrome frequently seen in chronic alcoholics. Cuinicat Cornecare \alieatid Beticiéhiey is most commonly Sey Peerancy id aicaioten. Deficiency states can result in mega- lest ane onder ie es Supplementation is recommen: ‘and dluring pregnancy. Cuinrcat Conecare amin Gdeficeny resis n exacted by poor bout a and bone formation, gum changes and petechise 345NBDE 1: BiocHEmIsTRY Cuimicat CoRRELATE A deficient af vitariniDloccurs with ack ‘of sunshine or renal failure, and leads to ‘ickets in chicren and osteomalacia in adults. Excessive amounts of vitamin D : Tamers inckeling proline and bsine hydrayiation For glimmer thesis'and dopamine hydroxylation in catecholamine synthesis. Vitamin C also serves as an antioxidant in cells. 9. Vitamin B12, or cobalamin, is synthesized exclusively by microorgan- isms but is conserved in animal tissues, where itis found in high con- centrations in the liver and kidney. It is required as the coenzyme for two reactions in human biochemistry: the methylation of homogys teine to methionine, and the conversion of methylmalonyl-CoA to succinylCoA. The absorption of vitamin By requires intrinsic factor, a protein synthesized by parietal cells of the gastric mucosa. A defi- ciency of vitamin B12 is rare, with the most common cause being a defect in absorption. A deficiency of vitamin B12 leads to mega- loblastic anemia, with or without.an.associated nervous.system neu- ropathy due to widespread demyelination. B. Fat-soluble vitamins 1. Vitamin A is supplied by several sources, including yellow and orange vegetables, liver, milk, and eggs. Vitamin A exists in three forms: retinal, retinol, and retinoic acid. Retinal acts as a cofactor for the protein opsin to form a rhodopsin complex, which acts as the light receptor in the visual process. Retinol andifetinoicjacid are required for growth, differentiation, and maintenance of epithelial cels; they bind to. nuclear receptors and regulate, the rate of transcription of specific genes. Patients with fat malabsorption or celiac disease may iGesomettitsrnin Aldeficient) producing hight blindnear and chymest of the conjunctiva, cornea, and skin (hyperkeratosis). Excessive amounts of this vitamin are also toxic to humans, producing joint pain, headache, and long bone thickening. 2. Vitamin D is found in high concentrations in fish oils, liver, and forti- fied milk. It can also be synthesized in human skin by ultraviolet irra diation of sterols. The vitamin is metabolically activated by sequen- tial hydroxylation in the liver and kidney to produce the active form Cf the vitamin, ,254{OH)z-Vit/D. One of the major roles of vitamin D {s to increase intestinal calcium and phosphate absorption. It binds to nuclear receptors and increases the rate of transcription of the gene coding for a protein that transports calcium from the lumen into the Intestinal mucosal cell. It also acts in concert with parathyroid hor- mone to mobilize calcium from bone. 3. Vitamin K is synthesized by intestinal bacteria and is supplied by leafy green vegetables. Vitamin K acts as a coenzyme for glutamate car- onylase, an enzyme that catalyzes the carboxylation of glutamic acid side chains in several ofthe clotting factors (factors li, Vl, and X). A deficiency of vitamin K results in an accumulation of pre thrombin, a deficiency in prothrombin, and an areeiaieces time, Newborns are vitamin K deficient since their intestinal tract is KAPLAN MEDICALPROTEINS, ENZYMES, AND COENZYMES e stil sterile, Antibiotic sterilization of the gut or malabsorption of fat ‘can lead to deficiency and bleeding complications, 4. Vitamin E, also known asst@eopherGl!’is found in leafy vegetables, vegetable oils, and grains. The major function of vitamin E is as an antioxidant, where its first line of defense is against peroxidation of polyunsaturated fatty acids found in cellular membranes. Fat malab- sorption may lead to vitamin E deficiency. In newborns, symptoms include hemolytic anemia; in adults, sensory ataxia due to spinocere- Gali deedguemina we) Sect humor rare Gacy seen frequently in premature infants and malabsorption syndromes. KAPLAN MEDICAL 347Bioenergetics > 2 ‘Coisextrtlenergyifrom f68d by the oxidation of carbohydrates, proteins and fas to.COp and HzO. catabolic or degradative path- ways involve oxidation reactions that release energy, most of which is captured in the high-energy phosphate bonds of ATP. In contrast, anabolic (synthetic) pathways involve reduction reactions that require the input of energy, with the reducing power and energy being supplied by NADPH and ATP, respectively. The pathways for catabolism of the metabolic fuels « a com- mon intermediate in the degradation of carbohydrates, proteins, and fats, AGSIVHEBANs oxidized in the mitochondria. va the citric cle. The released energy is conserved as electron pairs that are transferred from acetyl-CoA to NADH and FADH The electrons are then transferred sequentially from NADH and FADH, to Op, of H20. The oxidation of NADH and FADH2 by molecular oxygen, a process catalyzed by the electron transport chain, is a highly exergonic process. The energy released {s used to drive the phosphorylation of ADP to form ATP, an ender- gonic reaction. Most of the ATP. supply. in the ceil is derived from ‘oxidative phosphorylation. The regulation of oxidative phosphory- lation and the citric acid cycle are closely linked—they are both dependent upon the availability of molecular oxygen. This chapter will review the principles of thermodynamics and oxidation-reduc- tion reactions that form the basis for energy metabolism. cron resulting in the form: METABOLIC SOURCES OF ENERGY oy EE See eed ctr rs pris Son a fur anor gre 31. {GRRBMIEB tsb fuels ere yarotyeed to a chverse set of monomeric ‘BUIIBIRGIbI6eks, including glucose, amino acids, and fatty acids. In thee KAPLAN MEDICAL 349NBDE 1: BlocHemistry 350 nd staBh, the building blocks are degraded by various pathways to a com @ metabolic intermediate. (@eetykG@AY Most of the energy contained in ‘metabolic fuels is conserved in the chemical bonds (electrons) of acetyl-CoA In $t2G6 thE, thereitrieadld (Krebs, or tricarboxylic acid [TCAI) cycle ox!- dizes acetyl-CoA to.COz, and the electrons pairs present in the carbon-car- bon and carbon-hydrogen bonds are transferred to the electron carriers, NADH and FADHp. The ifialistage JA the extraction of energy from food is oxidative phosphorylation, where the energy in the electron pairs of NADH and FADH2 Is released via the electron transport chain (ETC) and is used to synthesize ATP, Stage Carbohydrate Protein Fat 1 Glutose Aming Acids Fatty Acids ae ; Pate ste ae ee Mw Se ve 2C02 e ankon @rADH, ¥ & 2 a N_.H,0 ATP +® AIP Figure 3-1. Extraction of energy from metabolic fuels. THERMODYNAMIC PRINCIPLES The principles underlying energy metabolism are based on the thermody- namics of chemical reactions. Some of the variables involved are: G = free energy (energy available to do work) anthalpy (heat content of a compound) $= entropy (randomness of a system) T = absolute temperature (measured in °K) R= gas constant (1.987 ca/moledegree) F = Faraday’s constant (23 kcal/voltemo!) KAPLAN MEDICAL> a BIOENERGETICS e ‘A. Free energy change (AG) of reactions. The free energy change of a sys- | ‘em is the portion of the total energy that is available for.usefull work. | For any chemical reaction, the AG is equal to is. The free energy change pre- dicts the direction in which a reaction will proceed spontaneously. IG = AH-TAS = Gproducts ~ Greactants The standard free eneFSYERANGEN(AG®) is a constant for any given reac- n. (The superscript indicates “standard conditions,” which are pH 7, and all reactants and products at 1.0 M concentration) The 4G? is elated to the equilibrium constant, Keg. For the reaction A+B —» C+D ‘the equilibrium constant is defined as: {cl {D1 Kea = [al (8) a6? “AT In Keg = -2:3 RT log Keq | Therefore, ifthe concentration of reactants and products at equilibrium are known, the Keq and the AG® for the reaction can be calculated, B. Exergonic and endergonic reactions are distinguished on the basis of whether the AG of the reaction is positive or negative. Exergonic reac- Iw a NursHete tions have a negative AG, and will proceed spontaneously in the direc- 2 AG 20-5 pny Se tion written. In contrast, endergonic reactions have a positive AG and = AG>0-> nor-spontaneods reaction require the input of energy to proceed in the direction written. If AG 0 equilibrium the reaction is at equilibrium, and the rates of the forward and the reverse reactions are equal. C. Coupled reaction systems. Endergonic reactions in metabolism frequent- ly proceed by being coupled to an exergonic reaction. The requirement for 2 coupled reaction system is that the product of the first reaction must be the substrate for the second,reaction. Many enzyme-catalyzed reactions that use ATP are examples of coupled reactions, where the ‘energy released by the hydrolysis of ATP is used to drive an endergonic reaction. For example, consider the phosphorylation of glucose to glu- cose-6-phosphate, a reaction that occurs in all cells and is catalyzed by hexokinase. Glucose + Pj —r Glucose-G-phosphate AG?= +3.3 kcal/mol KAPLAN MEDICAL 351NBDE 1: BlocHeMistaY Nore able reactions with ATP hydrolysis to Celis often couple energetically unfavor- | force the reaction to proceed. ATP + HO ——> ADP +P; Glucose + P| ——» Glucose-6-phosphate Sum: Glucose + ATP ——> Glucose-6-phosphate + ADP 352 —~ —< ee ee, direction of glucose-6-phosphate formation unless eneray is supplied. However 1 the eumtiche ol alusasnaiahalaals saul, t2 tre hydrolysis of ATP the sum of the. two.reactions is exergonie. The function of f@GKINGEE) therefore, is to couple these two reactions tat he enon ct lication favorable. D. Central role of adenine nucleotides in energy transduction. Some of the ‘common phosphate-containing compounds found in cells and the energy released by hydrolysis of their phosphate bonds under standard condi- tions are shown in Table 3-1. Table 3-1. Energy of hydrolysis of phasphate compounds Compound 8G° (kcal/mol) Phosphoenolpyruvate 148 1,3-Bisphosphoglycerate 1.8 Creatine phosphate “10.3 Pyrophosphate ATP. Glucose-1-phosphate Fructose-6-phosphate AMP Glucose-6-phosphate The positioning of these compounds in the table illustrates important con- cepts in energy transfer reactions. Note the structure of ATP: ° ° ° eee Aderine-ibose —0 ¢# 0, #0 oH! ¢ (¢ /¢ ester bond. (low energy) "anhydride bonds {high energy) Figure 3-2. Structure of ATR KAPLAN MEDICAL‘The energy inthe terminal anhydride bond of ATP is greater than the energy found in the compounds listed below it inthe table, and, there fore, can be used to drive the synthesis ofthese compounds. Conversely, the compounds listed above ATP have more energy in their phosphate bonds than does ATR, thus allowing these compounds to transfer ther phosphate groups to ADP with the formation of ATP. The conversion of ‘ADP to AP.by the use of high-eneray phosphate, metabolites is known 2s substrate-level phosphorylation As shown in Table 3-1, there are three phosphorylated intermediates in cells with sufcient anergy to par- ticipate in substrate-level phosphorylation. PROSpHOENOpyFuVEt® and ‘itbisphosehOGIjestatelare intermediates in glycols phosphate serves asa reservoir of high-energy phosphate bonds in mus cle. colysis, and relating) E, Other high-energy carriers. Many chemical groups that are transferred in metabolic reactions require “high- s" for the reaction to be exergonic, Examples of some groups that are transferred, their high- ‘energy carriers, and the types of reactions and/or pathways in which they participate are summarized in Table 3-2. Table 3-2, High-energy carriers of chemical groups in metabolism. Group Carrier Pathways/Reactions Pi are kinase reactions Sugars upP-suger Polysaccharide synthesis Acetate Acetyl-CoA. Fatty acid synthesis Fatty acids Acy-CoA, ‘TFiacylglycerol synthesis ‘Amino acids AMP.amino acid Protein synthesis Methyl SAM’ Methylation reactions | arbor! Catboxy-biotin Carbonylation reactions Sulfate Paps! Sulfation reactions *Seadenosyimethionine; 'Phosphoadenosine phasphsuifate OXIDATION-REDUCTION REACTIONS. ‘The principles of oxidation and reduction are an integral part of energy conservation in cells. The energy contained in metabolic fuels is released through a series of oxidation-reduction (F@H63) reactions that occur mainly in the mitochondria. Approximately fortyipercent of this energy is con- served as ATP, oes ‘A. General principles. Oxidation-reduction reactions involve the transfer of electrons between a donor and an acceptor. KAPLAN MEDICAL BIOENERGETICS ATP formation via substratelevel pphosphoriation occurs with ‘+ Phosphoenolpyrwvate 1 Taohnphogherate } 90% * Creatine phosphate -> muscle LEO the lion says GER: loss of electrons = oxidation ‘gain of electrons = reduction UL RIG: oxidation Is Joss (of electrons) reduction is gain (of electrons) 353NBDE 1: BiocHEMistrY Nore If the reduction potential is given, reverse the sign to find the oxidation potential: P= ~0.32 > Poe = 40,32 354 1. Defi eduction is defined as the gain of electrons. Every oxidation is accompanied by a reduction, each of which is considered to be a half- reaction. 2. The standard reduction potential, E°, is a constant that describes the tendency of a compound to be reduced. tis expressed in volts and is measured under standard conditions defined as 252GuipH) and at concentrations of 1.0 M for electron donors and acceptors. Values of standard reduction potentials for some common half-teactions are shown in Table 3-3. StrOngerielectronidoriors have a more negative reducti . Thus, under standard conditions, NADH with an €° of -0.32 volt will reduce pyruvate (or any other compound with a less negative E°), Table 3-3. Standard reduction potentials for common redox palr.* Half-Reaction EX) NADY +2074 HY ——> aH -032 Pyruvate +2e"+ 2H =» Lactate -019 Oxaloacetate + 2e"+2H* » ———» Malate “017 FAD + 2e° + 2H" ——> oH, -0.06 CoQ + 2e" + 2H" ———> coat, 40.10 Fumarate+2e"+2H* = > Succinate 40.13 | cyte (Fe) +e ——>_ Gaffe 40.29 12.02 + 2+ 2H+ —— > no 40.82 “Note that all ofthe reactions are written as reductions. 3. Relationship between AE? and AG®. The change in the standar reduc- tion potential for an oxidation-reduction reaction is related to the stan- dard free energy change (4G) for the reaction by the expression shown below, where n is the number of electrons transferred, F is the Faraday constant, and AE? = E°electron acceptor — electron donor. AG? = -nFae? From this relationship, itis clear that for an oxidation-reduction reac- tion to be exergonic and to proceed spontaneously, AE? must be a positive value. 2 Example. These thermodynamic principles can be illustrated by consider- ing the transfer of electrons from NADH to molecular oxygen. In this ‘example, NADH is the electron donor (it has the more negative standard reduction potential) and oxygen is the electron acceptor. The overall oxi- dation-reduction reaction can be written as the sum of two half reac- tions. KAPLAN MEDICAL