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Enzyme Specificity and Selectivity Introductory article Article Contents . Substrate Specificity Lizbeth Hedstrom, Brandeis University, Waltham, Massachusetts, USA . Stereospecificity . Reaction Specificity Specificity distinguishes enzymes from ordinary chemical catalysts. Specificity is evident in both discrimination between substrates and control of reaction outcome. Specificity arises from the three-dimensional structure of the enzyme active site. Substrate Specificity Enzymes have two extraordinary properties: (1) they are very efficient catalysts, accelerating reactions by as much as 1017-fold while operating in water, at neutral pH and ambient temperatures; (2) they are exquisitely selective, being capable of discriminating between closely related substrates and controlling reactions to yield a single product. While it is often convenient to consider these two properties separately, it is important to realize that they are inextricably intertwined: specificity is expressed in the rate at which a substrate is transformed to product. Enzyme specificity is measured by the value of kcat/Km Three Michaelis–Menten parameters describe a typical enzyme reaction: kcat, Km and kcat/Km. The kcat value is the turnover number; it measures the amount of product formed per enzyme molecule when all of the enzyme has bound substrate. It is a complicated kinetic constant that includes the rate constants for all the steps of the reaction after substrate binding. For example, kcat 5 k2k3/(k2 1 k3) for the relatively simple reaction of eqn [I]. Since some selection may occur during substrate binding, kcat is an inadequate measure of substrate specificity. Km is also a complex kinetic constant; it is an apparent dissociation constant of all enzyme-bound substrate complexes. For eqn [I], Km 5 k3(k 2 1 1 k2)/[k1(k2 1 k3)]. Like kcat, Km is also an inadequate measure of specificity: it does not take into account the rate of substrate turnover. In contrast, kcat/Km provides an accurate measure of specificity. This parameter is an apparent second-order rate constant for the reaction of free enzyme and free substrate. Since different substrates will compete for free enzyme, a comparison of the values of kcat/Km describes specificity. Not surprisingly, kcat/Km is also known as the specificity constant. Note that for eqn [I], kcat/Km 5 k1k2/(k 2 1 1 k2). This expression includes all of the steps up to and including the first irreversible step, k2 in this case. These are the only steps that determine specificity – once a substrate . Summary completes the first irreversible step, it is committed to form product and no further discrimination can occur. k9 k: k< 7 8 )* 78 ! 7; ! 7 ; k = 9 The reactions of serine proteases illustrate enzyme specificity The specificity of enzyme catalysis is best appreciated in contrast with chemical catalysis. For example, when peptide hydrolysis is catalysed by acid, every peptide bond is hydrolysed and the products are single amino acids (Figure 1a). In contrast, serine proteases hydrolyse peptides at discrete sites. For example, trypsin hydrolyses peptide bonds adjacent to positively charged residues such as lysine and arginine. The value of kcat/Km for hydrolysis at positively charged residues is approximately 104 times greater than that for hydrolysis at other residues. Moreover, while l- and d-peptide bonds are hydrolysed with equal efficiency in acid, only peptide bonds comprising l-amino acids are hydrolysed by trypsin. Similarly, chymotrypsin hydrolyses peptide bonds adjacent to large hydrophobic residues such as tryptophan, phenylalanine and tyrosine, while elastase hydrolyses peptides at small aliphatic residues such as alanine and valine. Thus, the products of enzyme-catalysed peptide hydrolysis will vary greatly depending upon the enzyme utilized. The serine protease reaction has three steps, as shown in eqn [II]: formation of a noncovalent enzyme–substrate complex; formation of an acyl-enzyme intermediate with the active site serine (acylation); and hydrolysis of the acylenzyme intermediate (deacylation). (Note that eqn [II] is formally the identical to eqn [I]. Therefore the expressions for kcat, Km and kcat/Km in terms of individual rate constants are the same as described above.) The individual rate constants for these steps can be derived from steadystate kinetics or determined directly in pre-steady-state experiments. Comparison of the reactions of trypsin with substrates containing either lysine or phenylalanine ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 1 Enzyme Specificity and Selectivity Ala + Arg + Glu + 2Gly + Lys + Leu + Phe + Pro + 2Ser + Thr + 2Val + H /H2O Val-Leu-Gly-Ser-Lys-Ser-Phe-Val-Pro-Gly-Thr-Arg-Ala-Glu trypsin Val-Leu-Gly-Ser-Lys + Ser-Phe-Val-Pro-Gly-Thr-Arg + Ala-Glu (a) L -Peptide Transition state − + NH 3 N H R H N C NH O R H O C NH H N O NH R′ CO2 + NH 3 + N H O R C NH H O R′ NH 2 CH 2 CH 2 CH 2 CH 2 H − C NH CO2 NH 3 N H CH 2 CH 2 CH 2 CH 2 O − − CO2 H C N NH − O NH O NH CH 2 CH 2 CH 2 CH 2 H C RCO E+ NH Lys CO2 O R′ D-Peptide − CO2 + NH 3 N H CH 2 CH 2 CH 2 CH 2 O R′ H N C NH H NH NH No reaction C O H O R (b) Figure 1 The hydrolysis of peptide bonds. (a) Comparison of the acid-catalysed and trypsin-catalysed reactions. (b) The active site of trypsin. residues at the site of hydrolysis reveals that most of the substrate discrimination occurs in the acylation step. Trypsin does bind lysine-containing substrates with 10-fold higher affinity than that for phenylalaninecontaining substrates; this difference in binding affinity is not sufficient to account for the 104-fold difference in kcat/ Km. However, once bound, lysine-containing substrates 2 react 103-fold faster than the phenylalanine-containing substrates. Thus, enzyme specificity is quantitatively derived from discrimination in the rate of chemical transformation, not from discrimination in substrate binding. ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Enzyme Specificity and Selectivity O k1 E + RCONHR′ E•RCONHR′ k2 E O C R k −1 OH OH R′NH2 k3 HOH E + RCO2H [II] OH Enzyme–substrate interactions approximate those of a lock and a key The specificity of trypsin, chymotrypsin and elastase arises from the three-dimensional structure of their respective active sites. Although the overall structures of these proteases are very similar, each enzyme has an active site that is sterically and electrostatically complementary to its substrate. Both trypsin and chymotrypsin have a deep pocket at their active sites; the trypsin pocket contains a negative charge that can complement the positive charge of the substrate, while the chymotrypsin pocket is hydrophobic, thus accounting for the preference for hydrophobic substrates (Figure 1b). Elastase has a shallow pocket that can only accommodate small residues. The pockets are arranged such that the carbonyl group of an l-amino acid residue is positioned to interact with the catalytic serine, the NH group makes a hydrogen bond to a main-chain carbonyl on the enzyme, and the a-hydrogen fits snugly in the active site (Figure 1b). Thus, substrate specificity is determined by the accumulation of noncovalent forces: hydrogen bonding, steric, electrostatic, van der Waals and hydrophobic. These interactions are reminiscent of the relationship between a lock and key: hydrophobic parts of the substrate bind in hydrophobic pockets on the enzyme, negative charges of the substrate interact with positive charges on the enzyme, and so forth. The active-site structure can also account for the stereospecificity of the serine protease reactions (Figure 1b). A d-peptide can bind at the active site, with the side-chain occupying the pocket as in an l-peptide. The a-hydrogen must also occupy the same position as in the l-peptide: the other groups are too bulky to occupy this space. Therefore, the carbonyl group of a d-peptide will not be positioned adjacent to the catalytic serine, and the enzyme cannot catalyse the hydrolysis of the d-peptide bond. Serine proteases are good examples of how steric and/or electrostatic exclusion can determine substrate specificity: a substrate that is too big, or of the wrong charge, cannot enter the active site. Enzymes can also discriminate against smaller substrates even though they are not excluded from the active site. For example, chymotrypsin hydrolyses peptide bonds adjacent to phenylalanine residues much more rapidly than those adjacent to alanine residues, although the smaller side-chain of alanine is clearly not prevented from entering the active site. The lock and key analogy can also be used to understand such discrimination against smaller substrates. A lock is exactly complementary to its key; this alignment allows the key to move the tumblers inside the lock and engage the locking mechanism. In contrast, while a smaller key may fit inside the lock, it will not move the tumblers. Likewise, a phenylalanine side-chain fits snugly in the chymotrypsin binding pocket. This interaction aligns the peptide bond in the optimal orientation with the catalytic residues of the enzyme and the reaction will be rapid. Although the alanine side-chain can also enter the binding pocket, it will not be firmly bound like the phenylalanine side chain; therefore, the binding energy of the alanine side-chain is insufficient to precisely align the peptide bond, and the reaction will be slower. Enzyme active sites are complementary to the transition state of the reaction While the lock and key analogy is useful for understanding enzyme–substrate interactions, it is important to remember that an enzyme active site is not simply complementary to the substrate. Such an enzyme would merely stabilize the ground state of the substrate, not accelerate the reaction. Catalysis results from selective stabilization of the transition state. Therefore, the enzyme active site must be complementary to the transition state of the reaction. This complementarity to the transition state produces a corresponding destabilization of the ground state. As stated by J. B. S. Haldane, ‘The key does not fit the lock quite perfectly, but exercises a certain strain on it.’ In the serine protease example, the transition state will have a tetrahedral structure with negative charge developing on the carbonyl carbon (Figure 1b). This tetrahedral oxyanion is stabilized by hydrogen bonds to two mainchain amide NH groups. However, these same hydrogen bonds to the substrate activate the carbonyl, priming it for reaction. Thus these hydrogen bonds strain the substrate; this strain is compensated in the favourable interactions between the negatively charged pocket of trypsin and the positively charged side-chain of the substrate. This is an example of a phenomenon William Jencks has described as the Circe effect: favourable interactions with one part of the substrate pay for unfavourable interactions (i.e. destabilization of the substrate) at the site of chemical transformation. The favourable interactions are used to ‘lure’ the substrate into the active site; the destabilization promotes the chemical transformation. Such destabilization includes desolvation and conformational restriction as well as electrostatic interactions. For example, the energetic cost of removing a substrate from water is very high; therefore, the affinity of the substrate for the enzyme is weak. However, chemical reactions proceed much faster in the absence of water. Thus, the cost of desolvating the substrate earns a large payback in the acceleration of the ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 3 Enzyme Specificity and Selectivity reaction. The Circe effect explains why specificity is not simply determined by the affinity of substrate binding: the favourable enzyme–substrate interactions are expressed in reaction rate, not in substrate affinity. Enzymes can change conformation in response to their substrate The lock and key analogy falls short in another regard. Whereas locks and keys have rigid structures, both enzymes and substrates are flexible. Substrate conformation can adapt to fit the enzyme active site. For example, while a peptide may not be conformationally constrained in solution, it must assume a fixed, extended conformation when bound to trypsin. Likewise, enzyme conformation can change in response to substrate binding. Daniel Koshland first proposed this ‘induced fit’ hypothesis: the binding of substrate can convert enzyme from an inactive conformation into an active one, by orienting catalytic residues, structuring a binding site for a second substrate, or closing the active site to exclude water. The adaptation of the active site to the substrate provides another mechanism of substrate discrimination. Hexokinase provides a classic example of induced fit. This enzyme catalyses the transfer of the g-phosphate group from ATP to the 6-hydroxyl group of glucose. The reaction with glucose is 107-fold more favourable than that with water as measured by kcat/Km. ATP binds poorly to hexokinase in the absence of glucose. The g-phosphate of ATP is disordered in this complex; this disorder suppresses the reaction with water. When glucose binds, hexokinase assumes a more compact structure; glucose is buried inside the enzyme with only the 6-hydroxyl exposed. This desolvation will promote catalysis, and the conformational change also increases the affinity of ATP. In addition, this conformation activates ATP, presumably fixing the position of the g-phosphate. The activation of ATP is apparent when glucose is replaced with a nonreactive analogue such as lyxose. Since lyxose cannot react with ATP, the reaction with water is accelerated by default: the value of kcat/Km for the hydrolysis of ATP increases 800-fold. This is an example of substrate synergism: the binding of one substrate to the enzyme triggers a conformational change that activates the second substrate. Some enzymes utilize proofreading reactions to increase specificity A stringent requirement for substrate selection is found in DNA replication, where the incorporation of the wrong nucleotide can have catastrophic effects on cell replication. DNA polymerase makes errors at the astoundingly low frequency of one error for every 108 –1012 nucleotides. However, if substrate discrimination were based on Watson–Crick base-pairing interactions alone, DNA 4 polymerase would make errors at a rate of one in every 104 nucleotides. The high fidelity of DNA polymerase is achieved by two levels of substrate selection. First, a nucleotide triphosphate is incorporated into the growing DNA chain. In the event that a mismatched nucleotide is inadvertently added, an editing reaction occurs at a second active site that removes the mismatched nucleotide. The error frequency for the overall reaction is the product of the frequencies of each separate active site; that is, if each site makes one error in 104 nucleotides, the overall reaction will have one error in 108 nucleotides. A comparable editing mechanism is observed in the enzymes that attach amino acids to their cognate tRNAs. Stereospecificity Stereospecificity also distinguishes enzyme reactions from ordinary chemical catalysis. The stereospecificity of enzymes is most dramatically illustrated by the reaction of dehydrogenases. As first demonstrated by Frank Westheimer, these enzymes discriminate between the hydrogens at C4 of NADH even though C4 is a symmetrical centre. These hydrogens are prochiral: the replacement of HR with a deuterium will produce an R chiral centre, while the replacement of HS with a deuterium will produce an S chiral centre. Vernon Anderson has shown that HR is transferred in greater than 99.999 998% of the turnovers of lactate dehydrogenase (Figure 2). As in the case of the serine proteases, the stereospecificity of the lactate dehydrogenase can be rationalized from the threedimensional structure of the enzyme active site and the relative positions of the pyruvate and NADH binding sites (Figure 2): pyruvate is bound by interactions at its carboxyl and carbonyl oxygens, while the nicotinamide ring is oriented by the interactions of its carboxamide group. These interactions place HR adjacent to the carbonyl carbon of pyruvate, ideally positioned for transfer. In contrast, HS points away from pyruvate; clearly the transfer of HR will be preferred over HS. However, these interactions must prevent the occasional rotation of the nicotinamide, with subsequent transfer of HS. The energetic barrier for this rotation must be approximately 42 kJ mol 2 1 to account for the magnitude of the preference for HR. The structural basis of this discrimination remains a mystery. Reaction Specificity Chemical reactions rarely proceed with 100% yield of a single product: high-energy intermediates usually decompose via several pathways to generate several different products. In contrast, the high-energy intermediates in enzyme reactions routinely follow a single pathway to ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Enzyme Specificity and Selectivity OH H2 N Nicotinamide R O HS N (C4) HR H2 N O C C O H2 N C Pyruvate O CH3 HN NH H2 N N H NH2 C NH OH H2 N O R HS N H2 N O HR C O H2 N C N H C HO HN N CH3 H2 N C NH2 NH Figure 2 The lactate dehydrogenase reaction. achieve 100% yields of a single product. This phenomenon is illustrated by the reaction of triose-phosphate isomerase. This enzyme catalyses the interconversion of glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) via an enediol intermediate (Figure 3a). However, in solution the enediol intermediate is not protonated to form DHAP. Instead, the enediol decomposes to methylglyoxal via the elimination of phosphate. Methylglyoxal is not observed in the enzyme-catalysed reaction. The elimination reaction requires that phosphate assume a position orthogonal to the plane of the enediol. In this position, the C–O bond will have the maximum overlap with the orbitals of the double bond. The enzyme prevents the elimination reaction simply by holding the phosphate in the plane of the enediol. Thus, triose-phosphate isomerase controls the reaction of the enediol intermediate by constraining its conformation. The reaction of triosephosphate isomerase is an example of stereoelectronic control. The ability of enzymes to harness the versatility of pyridoxal phosphate and similar cofactors is another example of stereoelectronic control. Pyridoxal phosphate is required in a diverse set of reactions involving amino acids: racemization, decarboxylation, transamination, belimination/replacement and g-elimination/replacement (Figure 4a). Pyridoxal phosphate chemistry begins with the formation of an imine with the amino group of an amino acid (Figure 4b). The next step is generation of a carbanion at the a-carbon of the amino acid. This carbanion can be formed by extraction of the a-proton or by decarboxylation of the amino acid. The pathway for formation of the carbanion is controlled by the orientation of the substrate – as in the case of triose-phosphate isomerase, the departing group must be positioned orthogonal to the plane of the imine double bond. This carbanion will undergo further transformations, including protonation, deamination and elimination reactions. In solution, all of these reactions can occur, producing a mixture of products. However, the enzyme reactions are tightly controlled by the orientation of the substrate on the enzyme and by the positioning of acidic and basic residues in the enzyme active site. Thus, despite the reactivity of the pyridoxal phosphate cofactor, each enzyme catalyses a single chemical transformation. Perhaps the most striking example of stereoelectronic control is found in the biosynthesis of sesquiterpenes. These compounds contain 15 carbons that can be arranged in over 200 different carbon skeletons, some of which are shown in Figure 5. This varied array derives from a single precursor, farnesyl diphosphate, via the rearrangement of an allylic carbocation intermediate. While in solution such carbocation rearrangements produce a hopelessly complex mixture of compounds, the enzyme-catalysed rearrangements produce a single compound. The enzymatic rearrangement reactions are presumably controlled by the conformation of farnesyl diphosphate on the enzyme and the electrostatic surface of the enzyme active site. Summary Specificity is a hallmark of enzyme catalysis; it is inseparable from catalytic efficiency, the other hallmark of enzyme reactions. Specificity arises from the threedimensional structure of the enzyme active site; this site is complementary to the transition state of the reaction. The substrate fits snugly within the enzyme active site, ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 5 Enzyme Specificity and Selectivity O G3P H C C H H OH C OH Enediol C 2_ H CH 2OPO3 OH DHAP C H O C H 2_ CH 2OPO3 OH C 2_ OPO3 H Rotates in solution H C 2_ O3PO H H OH C Methyl glyoxal O O OH C OH C H C C C H H Pi C O CH 3 H Figure 3 The triose-phosphate isomerase reaction. R H – Racemization H C CO 2 R C + – CO 2 + NH 3 NH 3 CO 2 R Decarboxylation H R C CO 2– H C + NH 3 + – H – RCOCO2 RCH(NH 3 )CO 2 R Transamination H + NH 3 O – CO 2 C + NH 3 R C – CO 2 X β-Elimination/replacement H CH2X – CO 2 C +Y H CH 2Y – CO 2 C + + NH 3 NH 3 X γ-Elimination/replacement H CH 2CH 2X CO 2– C + NH 3 +Y H CH 2CH 2Y – CO 2 C + NH 3 (a) Figure 4 Pyridoxal phosphate chemistry. (a) Reactions of pyridoxal phosphate. (b) Stereoelectronic control of carbanion formation in pyridoxal phosphate reactions. 6 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Enzyme Specificity and Selectivity PPi Farnesyl diphosphate ⊕ OPPi Pentalenene synthase Trichodiene synthase H Bisabolene synthase Bergamotene synthase Aristolochene synthase and so on Figure 5 The biosynthesis of sesquiterpenes. optimally aligned to react with the catalytic residues. In addition, the conformation of the substrate is constrained, which will control the course of the reaction. Further Reading Cane DE (1990) Enzymatic formation of sesquiterpenes. Chemical Reviews 90: 1089–1103. Fersht AR (1985) Enzyme Structure and Mechanism, 2nd edn. New York: WH Freeman. Hedstrom L (1996) Trypsin: a case study in the structural determinants of enzyme specificity. Biological Chemistry 377: 465–470. Jencks WP (1980) Binding energy, specificity, and enzymatic catalysis: the Circe effect. Advances in Enzymology and Related Areas of Molecular Biology 43: 219–410. Knowles JR (1991) To build an enzyme.... Philosophical Transactions of the Royal Society London Series B 332: 115–121. Koshland DE (1958) Application of a theory of enzyme specificity to protein synthesis. Proceedings of the National Academy of Sciences of the USA 44: 98–104. ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 7