Enzyme catalysis: removing chemically ‘essential’ residues by site-directed mutagenesis

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Enzyme catalysis: removing chemically ‘essential’ residues by site-directed mutagenesis Alessio Peracchi Enzymatic catalysis relies on the action of the amino acid side chains arrayed in the enzyme active sites. Usually, only two or three ‘essential’ residues are directly involved in the bond making and breaking steps leading to product formation. For the past 20 years, enzymologists have been addressing the role of such residues by changing them into chemically inert side chains. Removal of an ‘essential’ group often does not abolish activity, but can significantly alter the catalytic mechanism. Such results underscore the sophistication of enzyme catalysis and the functional plasticity of enzyme active sites.

Alessio Peracchi Dept of Biochemistry and Molecular Biology, University of Parma, 43100 Parma, Italy. e-mail: [email protected]

Enzymatic reactions display impressive rate enhancements (generally >109-fold) compared to their nonenzymatic counterparts1. Enzymes carry out catalysis by binding and positioning their substrate(s) in an active site lined with amino acid residues that participate in the chemical transformation. The main functions of these residues are believed to be modulation of the electrostatic environment, facilitation of proton transfer reactions, and covalent chemistry at the reaction center. Until the 1970s, the only way to address the importance of individual side chains in catalysis was to modify them using chemical reagents. Although this approach was limited and often nonspecific, in some cases it allowed the identification of residues whose modification could drastically inhibit the enzyme and that were convincingly shown to be implicated in the catalytic mechanism2. The picture of enzyme catalysis that emerged from such experiments emphasized the role of the few ‘essential’ residues involved in the chemical reaction, sharply distinct from the rest of the enzyme side chains, which contributed at most to the integrity of the active-site structure (for example, see Ref. 3). Since ~1980, site-specific mutagenesis has allowed the replacement of any amino acid residue within a protein with any other, through modification of the corresponding gene and expression of the mutated protein in a suitable host cell4. Mutagenesis is more specific and less structurally disruptive than chemical modification. In addition, it can be used to test side chains for which no specific chemical reagent exists. Mutagenesis therefore represents an excellent and universally adopted technique by which to probe the importance of specific residues in enzyme catalysis5,6. There have been a multitude of site-directed mutagenesis studies of enzymes; this review focuses on http://tibs.trends.com

those in which catalytic residues known (or at least strongly suspected on the basis of structural and biochemical evidence) to act as general acids or bases, or as nucleophiles, have been changed into unreactive side chains. Understanding the effects that can be expected from such mutations could be particularly instructive. For example, these kinds of mutations are sometimes used to infer quantitatively the contribution of given residues to catalysis. Furthermore, replacing ionizable or nucleophilic amino acids with unreactive ones is a common strategy for identifying ‘essential’ residues in the absence of precise structural information (for example, see Ref. 7). General acids, general bases and catalytic nucleophiles, arguably represent the most ‘essential’ residues in an active site as they directly participate in the formation and rupture of covalent bonds. However, this review provides a series of examples showing that mutation of these residues generally does not annihilate activity and sometimes even leaves most of the catalytic power of the enzyme intact6. Moreover, the article stresses that enzymes deprived of important catalytic groups perform catalysis through mechanisms that can be significantly modified with respect to the wild-type mechanism. Such altered mechanisms rely on the presence in the active sites of multiple catalytic devices and, sometimes, on the surrogate action of groups that do not participate in the wild-type reaction, blurring the distinction between ‘essential’ and ‘nonessential’ residues. Furthermore, the efficiency of the alternative mechanisms can depend heavily on the specific enzyme and on the type of amino acid substitution. Examples of mutations eliminating catalytic acids and bases

Table 1 reports the effects on kcat and Km of point mutations that eliminate ‘essential’ groups in a series of enzymes. The enzyme systems considered are structurally and functionally well characterized, and the mutations involve substitution of a side chain with either another of similar size (e.g. the replacement of Glu with Gln) or with a smaller one (e.g. the replacement of Glu with Ala). Such mutations are the least likely to grossly alter protein conformation2, a fact that facilitates interpretation of the experimental results2.

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Table 1. Examples of mutations eliminating catalytic acids or bases, or nucleophiles Mutant Ribonuclease T1

∆5-3-ketosteroid isomerase

Glucoamylase

Subtilisin

β-lactamase Fructose-2, 6-bisphosphatase

Role of mutated residue

kcat(s–1)a

µM) Km (µ

kcat/knonb

Refs

Wild type

–

310

134

1011

9

Glu58Ala

General base

11

85

4 × 109

9

Glu58Gln

General base

5.4

296

2 × 109

9

His92Ala

General acid

0.0022

144

9 × 105

11

His92Gln

General acid

0.173

255

7 × 107

11

Wild type

–

53 600

340

2 × 1011

15

Asp38Asn

General base

0.13

102

4 × 105

15

Asp38Ala

General base

400

122

109

16

Wild type

–

59.7

120

1012

19

Glu179Gln

General acid

0.047

150

8 × 108

18

Glu400Gln

General base

1

380

2 ×1010

19

Wild type

–

59

220

5 × 109

20

Ser221Ala

Nucleophile

3.4 × 10–5

420

3 × 103

20

His64Ala

General base

3.8 × 10–5

390

3 × 103

20

Wild type

–

2800

1000

9 × 109

24

Ser70Ala

Nucleophile

0.25

1300

8 × 105

24

Wild type

–

0.032

0.055

6 × 107

25

His256Ala

Nucleophile

5.5 × 10–3

0.88

107

25

Glu325Ala

General base

1.9 × 10–3

9.1

4 × 106

27

ak

and Km are the kinetic parameters most commonly reported in enzyme mutagenesis studies, and all the comparisons in this review are cat based on such parameters. It must be recalled that kcat reflects the rate(s) of the slowest step(s) of an enzymatic reaction2 and can be substantially slower than the rate of the chemical step. Thus, if a residue involved in chemistry is mutated but the chemical step is not ratelimiting for the wild-type enzyme, the impact of the mutation on kcat will be less than the actual impact on the chemical transformation. Furthermore, if a residue involved in both chemical and non-chemical steps is mutated, the impact on kcat will be a complex function of the impacts on all of the affected steps. bk represents the observed rate constant for the nonenzymatic conversion of substrate into product under solution conditions (e.g. pH, non temperature) analogous to those of the enzymatic assay1. There are many ways in which the rates of enzymatic and nonenzymatic reactions can be compared (for examples see Refs 1,44). The kcat/knon ratio is not more informative than other comparisons, but it is used throughout this paper because, intuitively, it mirrors the difference in reactivity of a substrate bound to the enzyme active site as compared to the same substrate free in solution (but see note 'a' above). The knon values reported here were obtained or estimated from the following works: ribonuclease T1 reaction, Ref. 45; ∆5-3-ketosteroid isomerase reaction, Ref. 47; glucoamylase reaction, Ref. 46; subtilisin reaction, Ref. 20; β-lactamase reaction, Ref. 48 and references therein; fructose-2, 6-bisphosphatase reaction, Ref. 49 and references therein.

Ribonuclease T1 from Aspergillus oryzae8 cleaves the RNA phosphodiester bond 3′ to G residues (Fig. 1), accelerating the process by 1011-fold compared to the nonenzymatic reaction (Table 1). There is little doubt that Glu58 and His92 serve as a general base and general acid, respectively, in the catalytic mechanism8–12. Mutating His92 to either Gln or Ala O N N

O N

NH N

NH2

N

O O –

O P O

N

H

O

NH N

NH2

O O H

O

O P

–

O

O

Glu58

H N

+

H His92

H O Glu58 O

O

HN

O–

O

N His92 Ti BS

Fig. 1. Mechanism for the transphosphorylation reaction catalyzed by ribonuclease T1 (Refs 8–12). The catalytic residues, Glu58 and His92, are shown in green. The protons that become exchanged in the course of the reaction are shown in red and blue. His92 serves as a general acid protonating the 5’ oxygen leaving group, whereas Glu58 acts as a general base and abstracts a proton from the 2’ hydroxyl internal nucleophile. One of the two resulting cleavage products contains a 2’,3’ cyclic phosphate, which is subsequently hydrolyzed by the enzyme.

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decreased catalysis by 103- and 105-fold, respectively. More strikingly, mutating Glu58 to Ala decreased catalysis by only 30-fold, although the stereospecificity of the reaction was profoundly altered with respect to that of the wild-type enzyme9,11,12. It was proposed that, upon removal of Glu58, a nearby residue (His40) acts as a surrogate base9. ∆5-3-ketosteroid isomerase13 catalyzes the isomerization of ∆5-3-ketosteroids to ∆4-3-ketosteroids through a stereospecific transfer of the 4β proton to the 6β position (Fig. 2). Mechanistic and structural data on the Pseudomonas testosteroni enzyme indicate that Asp38 serves as the catalytic base abstracting the 4β proton of the substrate13,14 (Fig. 2). Substitution of Asp38 with Asn reduced kcat by nearly 106-fold, although the mutant protein retained significant catalytic power15 (Table 1). Remarkably, the kcat for the Asp38Ala mutant was just 140-fold lower than the kcat for the wild-type enzyme, and 109-fold faster than the uncatalyzed reaction16. It was proposed that a second Asp residue (Asp99) functions as an alternative acid–base catalyst in the reaction mechanism of Asp38Ala (Ref. 16). Glucoamylase17 catalyzes the release of D-glucose from the nonreducing end of starch and related polysaccharides, with inversion of the anomeric

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(Dienolate intermediate)

H

O

–

O O

O

O

O

H

O

H –O

O

O

H

H

O

H H –

O

Tyr14

Tyr14

Tyr14

H

O

Asp38

Asp38

O

O

Asp38 Ti BS

Fig. 2. Mechanism of the ∆5-3-ketosteroid isomerase reaction13,14. Asp38 serves as the catalytic base, abstracting the 4β proton of the substrate 5-androstene-3,17-dione to form a dienolate intermediate. Subsequently, Asp38 transfers the proton to C6 of the intermediate to produce 4-androstene-3,17dione. Tyr14 is a catalytic residue whose hydroxyl group was formerly proposed to stabilize the dienolate intermediate-transition state through general acid catalysis15 and is now believed to act via formation of a strong hydrogen bond13,14. Activity of the mutant Tyr14Phe remains over a million-fold higher than background15.

configuration (Fig. 3). The enzyme from Aspergillus spp. accelerates the nonenzymatic reaction by an estimated 1012-fold (Table 1). The enzymatic mechanism is believed to directly involve two groups: Glu179 acts as a general acid donating a proton to the oxygen of the scissile bond, whereas Glu400 functions as a general base to activate a H2O molecule for the nucleophilic attack17. Whereas the Glu179Gln mutant showed a decrease in activity of >1000-fold18, the activity of the Glu400Gln mutant was only reduced 60-fold19. The reasons for the high residual activity of Glu400Gln are not understood, but it was hypothesized that electrostatic stabilization of the reaction’s transition state could be sufficient to allow efficient reaction even with an unactivated H2O molecule19. Examples of mutations eliminating catalytic nucleophiles

Subtilisin is a classic serine protease. In a series of studies that have become classics themselves20,21, Carter and Wells explored the importance of the residues in the catalytic triad of this enzyme. Even after removing Ser221 (the active-site nucleophile) subtilisin retained a kcat ~3000-fold higher than the uncatalyzed reaction; however, the reaction mechanism of the mutant enzyme must have changed, presumably to include direct attack of water on the Glu400 –

H HO

O

H

O

O

HO

H O

O

O HO

OH O

HO

Glu400

HO O

OH HO

O

Glu179

O

O H

HO –

O

HO

O

OH

HO

O

H

H O

HO

OH

O

Glu179 Ti BS

Fig. 3. Mechanism of hydrolysis of the α-1,4-glycosidic bond in D-glucose polymers catalyzed by glucoamylase17. Glu179 serves as a general acid protonating the leaving group oxygen, whereas Glu400 deprotonates a water molecule (shown in blue), favoring its nucleophilic attack on C1.

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peptide carbon20. The authors took pains to rule out most possible artifacts (Box 1). For example, they excluded the possibility that the residual activity of the Ser221Ala mutant was caused by contaminating wild-type subtilisin as the activity of their preparation was not affected by phenylmethylsulfonyl fluoride, a covalent inhibitor of serine proteases that reacts specifically with the serine nucleophile. Catalysis by the Ser221Ala mutant was therefore attributed to stabilization of the transition state of the hydrolysis reaction by noncovalent enzyme–substrate interactions20. Class A β-lactamases22 accelerate the opening of the β-lactam ring of penicillin antibiotics by ~1010-fold (Table 1). Here, too, the reaction mechanism proceeds via formation of an acylenzyme adduct between the substrate and a Ser residue (Ser70)22. Replacing Ser70 with Ala in β-lactamase from Bacillus licheniformis yielded a severely impaired mutant that, nevertheless, retained a kcat ~106-fold higher than background23. Analogously, the Ser70Ala mutant of the enzyme from Streptomyces albus could still accelerate β-lactam hydrolysis by nearly a million fold (Table 1), even in the presence of β-iodopenicillinate, a covalent inhibitor of β-lactamase that selectively modifies Ser70 in the wild-type enzyme24. The S. albus Ser70Ala mutant also showed a specificity towards different substrates that was markedly altered with respect to the wild-type enzyme24. These results suggested the existence of an alternative catalytic mechanism in the Ser70Ala mutants, involving the direct hydrolysis of the β-lactam amide bond23,24. Fructose-2, 6-bisphosphatase is part of a bifunctional regulatory enzyme (fructose-6phosphate, 2-kinase : fructose-2, 6-bisphosphatase) that controls the intracellular concentration of fructose 2, 6-bisphosphate25–27. The bisphosphatase reaction (fructose-2, 6-bisphosphate → fructose-6phosphate + inorganic phosphate) apparently proceeds through nucleophilic attack of His256 on the substrate, with formation of a covalent phosphohistidyl intermediate, which has been isolated25. Surprisingly, the replacement of His256 with Ala decreased kcat by less than one order of magnitude compared to the wild-type enzyme25. A series of controls argued against most possible artifacts. For example, inhibition experiments indicated that the residual bisphosphatase activity was not attributable to the kinase domain of the protein25. Activity of the His256Ala mutant appeared to depend on the presence of a second His (His390) in the phosphatase active site, but no covalent phosphorylenzyme intermediate could be isolated25. Based on these data and the His256Ala crystal structure, it was proposed that the mutant enzyme adopted an altered reaction pathway, involving direct nucleophilic attack of H2O on the phosphorus, assisted by general-base catalysis from the nearby His390 (Refs 25,26). The occurrence of such a reaction

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Box 1. Is the residual activity of impaired mutant enzymes an artifact? When dealing with the low residual activity of enzymes missing a catalytic group, before attempting any mechanistic interpretation, efforts must be made to exclude the possibility of artifacts, which are particularly insidious in these casesa,b. The weak activity (measured by steady-state kinetics) of a severely impaired mutant can sometimes arise from the presence of traces of wild-type enzyme in the preparation, either as a contaminant or because of natural errors of misincorporationa. In protein biosynthesis, the rate of misincorporation at a given position is in the order of 1 in 1000 (Ref. c), which therefore represents a rough upper limit for the frequency of accidental reinsertion of the wild-type amino acid at the mutated position. However, the limit is not strict and the actual frequency of reinsertion depends on many factors, such as the type and strain of the microorganism in which the recombinant protein is expressed and the codons used to specify the original and the substituted amino acidb–d. Note also that an enzyme mutated at a catalytic residue could regain activity because of a spontaneous chemical modification of the newly introduced side chain. This is possible, in particular when carboxylic residues (Asp and Glu) are replaced with their respective amides (Asn and Gln) – mutations that can be reversed by spontaneous hydrolytic deamidatione. Traces of wild-type enzyme present in a totally inactive mutant preparation would result in a lower kcat (directly proportional to the concentration of wild-type enzyme) and the same Km value as for the wild-type enzyme. A mutant showing a low kcat and an unaltered Km is, therefore, very suspiciousa. Contamination by wild-type enzyme can be reasonably excluded when a careful kinetic characterization of the mutant shows, for example, an altered dependence of activity on pH or a substrate specificity markedly distinct from that of the wild-type enzyme.

Even when the kinetic behavior of the mutant enzyme deviates significantly from that of the wild type, the presence of an active impurity cannot be completely ruled out because the contaminant could be a partially active mutant bearing some compensatory mutation (for examples, see Ref. f) or an unrelated enzyme (for example, a metalloprotease contaminating a preparation of serine protease mutant). Presumably, one should worry about second-site revertants only when dealing with extremely impaired enzymes (e.g. when a mutant preparation shows an activity ≤105-fold lower than wild type), both because of the above mentioned error rate in protein synthesis and because revertants are generally much less active than the wild-type enzyme. However, this cannot be taken for granted. As for heterologous contaminating enzymes, their presence can sometimes be suspected when the same mutant, purified through different protocols, shows substantially different specific activities. Elimination of heterologous contaminant activities requires rigorous purification procedures, although even these can be insufficient in some cases. References a Fersht, A. (1999) Structure and Mechanism in Protein Science – A Guide to Enzyme Catalysis and Protein Folding, Freeman & Co. b Schimmel, P. (1989) Hazards of deducing enzyme structure–activity relationships on the basis of chemical application of molecular biology. Acc. Chem. Res. 22, 232–233 c Freist, W. et al. (1998) Accuracy of protein biosynthesis: quasi-species nature of proteins and possibility of error catastrophes. J. Theor. Biol. 193, 19–38 d Stansfield, I. et al. (1998) Missense translation errors in Saccharomyces cerevisiae. J. Mol. Biol. 282, 13–24 e Pries, F. et al. (1995) Activation of an Asp124>Asn mutant of haloalkane dehalogenase by hydrolytic deamidation of asparagine. FEBS Lett. 358, 171–174. f Yanofsky, C. et al. (1993) Partial revertants of tryptophan synthetase α chain active site mutant Asp60>Asn. J. Biol. Chem. 268, 8213–8220

pathway is still awaiting confirmation from stereochemical experiments26. Point mutations and modified mechanisms in enzyme active sites

The examples above confirm that even mutations removing bona fide catalytic groups fail, in general, to abolish the catalytic power of enzymes, implying that enzymatic activities do not necessarily rely on truly indispensable groups. This conclusion, which was not obvious before the advent of site-directed mutagenesis6, can be somewhat counterintuitive: for a reaction in solution that is, for example, general-base catalyzed, if no base is provided, no catalysis is observed. Furthermore, it might be expected that, for a reaction requiring nucleophilic attack of the catalyst on the substrate, catalysis should not occur in the absence of the catalyst’s nucleophilic functionality. But enzymatic reactions display a level of sophistication far superior to nonenzymatic reactions. Enzyme active sites have been designed by evolution to work as cooperative entities2,5,28 in which acid–base catalysis or even covalent catalysis is only part of the effort to lower the free energy of activation of a http://tibs.trends.com

reaction. In each active site, different mechanisms, besides the direct involvement of side chains in the chemical reaction, collaborate to achieve this goal2. Recognized strategies for catalysis include electrostatic and geometric complementarity to the transition state, desolvation and possibly distortion of the substrate, and use of binding energy for precise positioning and for lowering the entropy of activation2,28; these factors can have a substantial importance in determining the overall rate enhancement. For example, it has been shown that simply bringing together and correctly positioning two reactants in an enzyme active site, can provide a rate acceleration of up to 108-fold compared to 1 M reactants free in solution28. In the presence of multiple factors contributing to catalysis, mutation of just one catalytic device can simply favor the occurrence of a reaction through a slightly altered pathway or mechanism5. In particular, it can be relatively easy for an enzyme, upon loss of a catalytic general acid or base, to maintain some significant activity because a vicinal residue takes over the function of proton donor or acceptor9,16. In some cases, there is evidence that even ionizable groups on the substrate can be recruited as

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surrogate acids or bases, giving rise to so-called substrate-assisted catalysis29. These observations, and the fact that the chemical transformation does not necessarily represent the rate-limiting step for an enzymatic reaction (see footnote ‘a’ in Table 1), can explain why the removal of groups identified as catalytic acids or bases sometimes produces surprisingly modest effects on kcat (Refs 9,16,19,27). Moreover, it is plausible that a hydrolytic enzyme deprived of a catalytic nucleophile can accelerate the direct hydrolysis of the substrate20,23–25. The transition state for attack of, for example, a Ser hydroxyl on an electrophilic center, is presumably similar to the transition state for attack of H2O on the same center; thus, the noncovalent interactions that stabilize the former could also stabilize the latter20,21. In addition, substitution of Ser with Ala can create extra room in an active site, facilitating the access of solvent water. Consider also that, in some classes of hydrolases, only slight differences apparently separate enzymes that exploit covalent catalysis from others that do not. For example, phage T4 lysozyme, which usually does not form covalent intermediates, can be made to adopt covalent catalysis simply by introducing a His residue at a specific position in the active site30. Removal of a catalytic nucleophile is necessarily more difficult to offset for enzymes that employ a ping-pong mechanism, in which a covalent adduct mediates the transfer of a chemical group from a donor to an acceptor substrate (other than H2O). One such enzyme is nucleoside diphosphate kinase, which interconverts nucleoside diphosphates (NDPs) and triphosphates (NTPs) in two steps. First, a phosphoryl group is transferred from an NTP (donor) to the nucleophilic His122 in the active site; second, the phosphoryl group is transferred from the phosphohistidyl intermediate to an NDP acceptor. In the absence of the catalytic His, direct phosphoryl transfer from an NTP to an NDP would require simultaneous binding of both substrates to the active site, which is sterically implausible. Nevertheless, the His122Ala and His122Gly mutants of nucleoside diphosphate kinase possessed the ability to directly transfer the phosphoryl group from ATP to smaller acceptor substrates, including H2O (i.e. the mutants showed hydrolase activity) and, in the case of His122Gly, to a variety of alcohols and amines31,32. This example and others in the literature (see Refs 33,34 for examples) indicate that removal of an ‘essential’ group can change not just some aspects of the catalytic mechanism but also the type of reaction altogether. This behavior seems related to the phenomenon of ‘catalytic promiscuity’; that is, to the ability of enzyme active sites to carry out distinct chemical transformations, albeit with different efficiencies, provided that the transition states for such reactions are sufficiently analogous and can therefore be stabilized by the same catalytic machineries35. Removal of an active-site residue could substantially alter the balance between different http://tibs.trends.com

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reactions, favoring one activity over another. Catalytic promiscuity presumably plays an important role in the development of novel enzymatic activities during the course of evolution35. How important is an ‘essential’ residue?

The observation that mutating a catalytic group will alter, to some degree, the catalytic mechanism, limits the possibility of quantitating the contribution of an individual residue to catalysis by comparing the activities of the mutant and wild-type enzymes. Indeed, it has been stressed that the observed effect of a mutation inherently depends on the ‘context’ in which the mutation is introduced and on the identity of the substituting residue32. This remark applies to the effect of mutagenesis on many properties of a protein (such as, for example, its thermodynamic stability), but it seems particularly appropriate when dealing with catalysis6,32. The multifactorial and cooperative nature of catalysis represents, by itself, a fundamental ‘context’ effect. As the active sites of enzymes integrate the workings of multiple catalytic devices2,5,28, the actual importance of a given residue depends not only on the physical contacts it makes (i.e. the structural context), but also on its functional interconnection with the other catalytic devices6,10,21. For example, in subtilisin, as in most serine proteases, hydrogen bonds between the carbonyl oxygen of the cleavable peptide bond and amide groups in the so-called oxyanion hole are believed to contribute to stabilization of the transition state. Consistent with this, removal of one amide group from the oxyanion hole of wild-type subtilisin decreased catalysis by nearly 150-fold21. However, removal of the same group in the Ser221Ala mutant favored catalysis, increasing kcat by about sevenfold21. Small variations in both the structural context and the functional interplay between catalytic devices could explain why the same mutation can have disparate impacts in homologous enzymes6,36. For example, replacement of Ser70 with Ala in the β-lactamases from B. licheniformis and S. albus decreased catalysis by similar extents (~104-fold), but the same mutation was substantially more deleterious for the Staphylococcus aureus enzyme (≥105-fold decrease in activity37). The impact of removing an essential residue also depends on the specific mutation introduced, as illustrated by two examples in Table 1. In ribonuclease T1, changing His92 to either Gln or Ala effectively eliminates an important acid–base catalyst; yet the His92Gln was 80-fold more active than His92Ala (Ref. 11). Conversely, in ketosteroid isomerase, replacement of Asp38 with Asn was 2600-fold more deleterious than replacement with Ala (Refs 15,16). Thus, the type of amino acid substitution can largely determine the experimental readout, in ways that can be difficult to predict. This ambiguity can be alleviated by systematic studies in which the

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Acknowledgements I am grateful to Dan Herschlag, Suzanne Admiraal, Pat O’Brien, Geeta Narlikar, Riccardo Percudani and Andrea Mozzarelli for discussions and critical reading of the manuscript. I also acknowledge financial support from the Italian National Research Council – Target Project on Biotechnology.

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effects of different mutations at the same position are compared. Such an approach is potentially very informative, and further information can be gained through the use of particular mutagenetic techniques that allow the introduction of ‘unnatural’ amino acids into the active sites of enzymes38. The use of an expanded set of amino acids affords a detailed verification of the properties of a given side chain, such as, for example, size and pKa, that are most crucial for catalysis (see Ref. 39 for an example). In addition to these intrinsic limits of mutagenesis, more trivial reasons can increase the uncertainties in quantitating the contribution of a residue to the overall catalysis. For very impaired mutants, the presence of contaminants in the preparation could lead to underestimation of the effect of a mutation (Box 1). Also, if a mutant catalyzes a novel chemical transformation, this could prevent measurement of its residual activity in the ‘physiological’ reaction. Finally, the observed impact of a mutation on kcat depends on whether the mutated residue contributes to the rate-limiting step of the reaction (see footnote ‘a’ in Table 1). For example, it has been suggested that, in the reaction catalyzed by glucoamylase, product dissociation is slow and limits the rate of the overall catalytic process40. In that case, the observed effects on kcat of the mutations Glu179Gln and Glu400Gln (Table 1) would represent only lower limits for the actual impact of these mutations on the rate of chemical hydrolysis. This kind of uncertainty can only be avoided through a rigorous kinetic analysis of the reactions catalyzed by wild-type and mutant enzymes, including experiments (e.g. isotope

References 1 Radzicka, A. and Wolfenden, R. (1995) A proficient enzyme. Science 267, 90–93 2 Fersht, A. (1999) Structure and Mechanism in Protein Science – A Guide to Enzyme Catalysis and Protein Folding, W.H. Freeman 3 Lehninger, A.L. (1975) Biochemistry (2nd edn), Worth Publishers, New York 4 Smith, M. (1982) Site directed mutagenesis. Trends Biochem. Sci. 7, 440–442 5 Knowles, J.R. (1987) Tinkering with enzymes: what are we learning? Science 236, 1252–1258 6 Plapp, B.V. (1995) Site-directed mutagenesis: a tool for studying enzyme catalysis. Methods Enzymol. 249, 91–119 7 Hyun-Ju, K. et al. (2000) Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by sitedirected mutagenesis. FEBS Lett. 483, 139–142 8 Steyaert, J. (1997) A decade of protein engineering on ribonuclease T1. Atomic dissection of the enzyme substrate interactions. Eur. J. Biochem. 247, 1–11 9 Steyaert, J. et al. (1990) Histidine-40 of ribonuclease T1 acts as a base catalyst when the true catalytic base, glutamic acid-58, is replaced by alanine. Biochemistry 29, 9064–9072 10 Steyaert, J. and Wyns, L. (1993) Functional interactions among the His40, Glu58 and His92 catalysts of ribonuclease T1 as studied by double and triple mutants. J. Mol. Biol. 229, 770–781 11 De Vos, S. et al. (1998) Dissecting histidine interactions of ribonuclease T1 with asparagine http://tibs.trends.com

12

13

14

15

16

17

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effect studies) aimed at clarifying the rate-limiting step(s) of such reactions. Conclusions

For the past 20 years, site-directed mutagenesis has enriched our knowledge of the mechanisms of enzyme action, revealing more and more of a fascinatingly complex picture. In particular, mutagenesis of residues involved in catalysis has highlighted the interplay between different catalytic devices and strategies, softening the distinction between ‘essential’ and ‘nonessential’ groups and demonstrating the functional plasticity of enzyme active sites. This, in turn, has offered clues as to the evolution of enzymatic activities and could be valuable for the rational design of enzymes endowed with novel functions41. Some limits of mutagenesis have also become apparent. For example, the technique cannot rigorously establish the contribution of individual residues to the overall rate enhancement. Furthermore, when mutagenesis is used to search for catalytic residues, especially in the absence of structural information, its results can be ambiguous: removal of catalytic groups does not necessarily give huge effects whereas residues associated with large effects when mutated might not be directly involved in catalysis42–44. Far from devaluing mutagenesis as an experimental tool, these observations emphasize once more the importance of a detailed biochemical and structural characterization of the wild-type and mutant enzymes to support the identification of catalytic residues, to define their specific roles and to better understand the structural and functional context in which they operate.

and glutamine replacements: analysis of double mutant cycles at one position. J. Mol. Biol. 275, 651–661 Loverix, S. et al. (2000) Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond. Chem. Biol. 7, 651–658 Pollack, R.M. et al. (1999) Mechanistic insights from the three-dimensional structure of 3-oxo-∆5steroid isomerase. Arch. Biochem. Biophys. 370, 9–15 Cho, H.S. et al. (1999) Crystal structure of ∆5-3ketosteroid isomerase from Pseudomonas testosteroni in complex with equilenin settles the correct hydrogen bonding scheme for transition state stabilization. J. Biol. Chem. 274, 32863–32868 Kuliopulos, A. et al. (1989) Kinetic and ultraviolet spectroscopic studies of active-site mutants of ∆5-3ketosteroid isomerase. Biochemistry 28, 149–159 Henot, F. and Pollack, R.M. (2000) Catalytic activity of the D38A mutant of 3-oxo-∆5-steroid isomerase: recruitment of aspartate-99 as the base. Biochemistry 39, 3351–3359 Sauer, J. et al. (2000) Glucoamylase: structure/function relationships, and protein engineering. Biochim. Biophys. Acta 1543, 275–293 Sierks, M.R. et al. (1990) Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from Aspergillus awamori. Protein Eng. 3, 193–198 Frandsen, T.P. et al. (1994) Site-directed mutagenesis of the catalytic base glutamic acid

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21

22

23

24

25

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400 in glucoamylase from Aspergillus niger and of tyrosine 48 and glutamine 401, both hydrogenbonded to the γ-carboxylate group of glutamic acid 400. Biochemistry 33, 13808–13816 Carter, P. and Wells, J.A. (1988) Dissecting the catalytic triad of a serine protease. Nature 332, 564–568 Carter, P. and Wells, J.A. (1990) Functional interaction among catalytic residues in subtilisin BPN′. Proteins 7, 335–342 Matagne, A. et al. (1998) Catalytic properties of class A β-lactamases: efficiency and diversity. Biochem. J. 330, 581–598 Hokenson, M.J. et al. (2000) Enzyme-induced strain/distortion in the ground-state ES complex in β-lactamase catalysis revealed by FTIR. Biochemistry 39, 6538–6545 Jacob, F. et al. (1991) Active-site serine mutants of the Streptomyces albus G β-lactamase. Biochem. J. 277, 647–652 Mizuguchi, H. et al. (1999) Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway. J. Biol. Chem. 274, 2166–2175 Yuen, M.H. et al. (1999) Crystal structure of the H256A mutant of rat testis fructose-6phosphate,2-kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities. J. Biol. Chem. 274, 2176–2184

Review

27 Sakurai, M. et al. (2000) Glutamate 325 is a general acid–base catalyst in the reaction catalyzed by fructose-2,6-bisphosphatase. Biochemistry 39, 16238–16243 28 Jencks, W.P. (1975) Binding energy, specificity and enzymic catalysis: the Circe effect. Adv. Enzymol. 43, 219–410 29 Dall’Acqua, W. and Carter, P. (2000) Substrateassisted catalysis: molecular basis and biological significance. Protein Sci. 9, 1–9 30 Kuroki, R. et al. (1999) Structural basis of the conversion of T4 lysozyme into a transglycosidase by reengineering the active site. Proc. Natl. Acad. Sci. U. S. A. 96, 8949–8954 31 Admiraal, S.J. et al. (1999) Nucleophilic activation by positioning in phosphoryl transfer catalyzed by nucleoside diphosphate kinase. Biochemistry 38, 4701–4711 32 Admiraal, S.J. et al. (2001) Chemical rescue of phosphoryl transfer in a cavity mutant: a cautionary tale for site-directed mutagenesis. Biochemistry 40, 403–413 33 Malet, C. and Planas, A. (1998) From β-glucanase to β-glucansynthase: glycosyl transfer to α-glycosyl fluorides catalyzed by a mutant endoglucanase lacking its catalytic nucleophile. FEBS Lett.440, 208–212 34 Sharath, A.N. et al. (2000) Reviving a dead enzyme: cytosine deaminations promoted by an inactive DNA methyltransferase and an S-adenosylmethionine analogue. Biochemistry 39, 14611–14616

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35 O’Brien, P.J. and Herschlag, D. (1999) Catalytic promiscuity and the evolution of new enzymatic activities. Chem. Biol. 6, R91–R105 36 Pohjanjoki, P. et al. (1998) Evolutionary conservation of enzymatic catalysis: quantitative comparison of the effects of mutation of aligned residues in Saccharomyces cerevisiae and Escherichia coli inorganic pyrophosphatases on enzymatic activity. Biochemistry 37, 1754–1761 37 Chen, C.C. et al. (1996) Structure and kinetics of the β-lactamase mutants S70A and K73H from Staphylococcus aureus PC1. Biochemistry 35, 12251–12258 38 Dougherty, D.A. (2000) Unnatural amino acids as probes of protein structure and function. Curr. Opin. Chem. Biol. 4, 645–652 39 Thorson, J.S. et al. (1998) Analysis of the role of the active site tyrosine in human glutathione transferase A1-1 by unnatural amino acid mutagenesis. J. Am. Chem. Soc. 120, 451–452 40 Natarajan, S.K. and Sierks, M.R. (1996) Identification of enzyme–substrate and enzyme–product complexes in the catalytic mechanism of glucoamylase from Aspergillus awamori. Biochemistry 35, 15269–15279 41 Cedrone, F. et al. (2000) Tailoring new enzyme functions by rational redesign. Curr. Opin. Struct. Biol. 10, 405–410 42 Shi, Z. et al. (1993) Mechanism of adenylate kinase. What can be learned from a mutant

43

44

45

46

47

48

49

503

enzyme with minor perturbation in kinetic parameters? Biochemistry 32, 6450–6458 Wang, W. et al. (1998) Ambiguities in mapping the active site of a conformationally dynamic enzyme by directed mutation. Role of dynamics in structure–function correlations in Escherichia coli adenylosuccinate synthetase. J. Biol. Chem. 273, 16000–16004 Axe, D.D. et al. (1998) A search for single substitutions that eliminate enzymatic function in a bacterial ribonuclease. Biochemistry 37, 7157–7166 Li, Y. and Breaker, R.R. (1999) Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2′-hydroxyl group. J. Am. Chem. Soc. 121, 5364–5372 Hirayama, F. et al. (1992) Acid-catalyzed hydrolysis of maltosyl-β-cyclodextrin. J. Pharm. Sci. 81, 913–916 Pollack, R.M. et al. (1989) Determination of the microscopic rate constants for the base-catalyzed conjugation of 5-androstene-3,17-dione. J. Am. Chem. Soc. 111, 6419–6423 Llinas, A. et al. (1998) Chemical reactivity of penicillins and cephalosporins. Intramolecular involvement of the acyl-amido side chain. J. Org. Chem. 63, 9052–9060 Kirby, A.J. and Varvoglis, A.G. (1967) The reactivity of phosphate esters. Monoester hydrolysis. J. Am. Chem. Soc. 89, 415–423