Portal de Periódicos da CAPES

Electrostatic Origin of the Catalytic Power of Enzymes and the Role of Preorganized Active Sites

1998; Elsevier BV; Volume: 273; Issue: 42 Linguagem: Inglês

10.1074/jbc.273.42.27035

1083-351X

Arieh Warshel,

Metal-Catalyzed Oxygenation Mechanisms

ground state transition state distant binding hydrogen bond low barrier hydrogen bond empirical valence bond. Enzymatic reactions are involved in most biological processes. Thus, there is a major practical and fundamental interest in finding out what makes enzymes so efficient. Many crucial pieces of this puzzle were provided by biochemical and structural studies (1Fersht A.R. Enzyme Structure and Mechanism. W. H. Freeman and Co., New York1985Google Scholar). Yet, as will be shown below, the actual reason for the catalytic power of enzymes is not widely understood. It is clearly not explained by the statement that “the enzyme binds the transition state stronger than the ground state” because the real question is how the differential binding can be accomplished. Similarly, it is not true that “evolution can use any factor to accelerate reactions.” This review uses energy considerations and the results of computational studies to clarify open questions about enzyme catalysis. Examples of the enormous catalytic power of enzymes have been eloquently compiled by Wolfenden (2Wolfenden R. Science. 1983; 222: 1087-1093Crossref PubMed Scopus (107) Google Scholar). As is clear from this compilation and from kinetic considerations (e.g. Ref. 1Fersht A.R. Enzyme Structure and Mechanism. W. H. Freeman and Co., New York1985Google Scholar), many enzymes evolved by optimizingk cat/K m. The energetics associated with the interplay between k cat and K m is considered in Fig. 1. As is obvious from this figure, enzymes can increase their rate by providing strong binding to the ground state (reducing K m) and reducing Δg cat‡ (increasingk cat). As will be emphasized below, the effect due to K m is quite clear and does not present a major puzzle. The real challenge is to find out what was used by evolution in increasing k cat. The fact thatk cat increases upon reduction of Δg cat‡ was basically stated by Pauling (3Pauling L. Chem. Eng. News. 1946; 24: 1375-1377Crossref Scopus (731) Google Scholar). However, the way by which Δg cat‡ is reduced was not addressed (except in a suggestion of a strain effect, which is now known to be rather small). Thus, the key question of how this reduction of Δg cat‡ is accomplished remained entirely unresolved. The studies of Jencks and co-workers (4Jencks W.P. Catalysis in Chemistry and Enzymology. Dover Publications, New York1986Google Scholar) provided a major insight by emphasizing the effect of binding energy and by trying to find out how an enzyme can reduce Δg cat‡. In the absence of other reasonable explanations (see below) it was concluded that Δg cat‡ is reduced by ground state (GS)1destabilization, which was attributed to entropic, strain, and desolvation effects (4Jencks W.P. Catalysis in Chemistry and Enzymology. Dover Publications, New York1986Google Scholar). These effects were invoked by many workers in the field (see below) and were fully consistent with the options available to organic chemists in catalyzing reactions in solution. Yet, more recent energy considerations have indicated that such effects are not likely to give large contributions to enzyme catalysis. The problems with GS destabilization mechanisms are illustrated by Fig. 1; increasing the GS energy without changing the TS energy will leave Δg ‡ unchanged. In other words, such changes will alter k cat and K m but leave k cat/K m constant (except in the diffusion control limit when k catis larger than k −1, which also supports our conclusion about GS stabilization 2Evolving to the case whenk −1 ∼ k cat cannot be accomplished by GS destabilization but by combining GS stabilization and TS stabilization.). Thus, an enzyme that was optimized under the evolutionary pressure of increasingk cat/K m would not gain much from changing its GS energy without changing the TS energy. To verify the above point, we introduce here a new framework for analyzing mutation experiments that should allow the reader to determine what wasreally done by evolution. To do so we classify mutation experiments into the three classes described in Fig. 2. If the processes of evolution lead to ground state destabilization, then going backward in evolution by mutations should lead to ground state stabilization, while leaving the TS energy unchanged. This will involve mutations that can be referred to as “GS mutations.” If an enzyme has evolved to stabilize its TS, then we will have mutations that can be referred to as “TS mutations.” If binding energy is used to stabilize the GS and TS by the same amount we will have mutations that can be referred to as “distant binding (DB) mutations” because such mutations are expected to operate by binding the substrate in regions that are far from its reactive part. An examination of mutations that reduce the activity of enzymes in a substantial way (5Leatherbarrow R.J. Fersht A.R. Winter G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7840-7844Crossref PubMed Scopus (158) Google Scholar, 6Radic Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (425) Google Scholar, 7Carter P. Wells J.A. Proteins Struct. Funct. Genet. 1990; 6: 240-248Crossref Scopus (111) Google Scholar, 8Ménard R. Plouffe C. Laflamme P. Vernet T. Tessier D.C. Thomas D.Y. Storer A.C. Biochemistry. 1995; 34: 464-471Crossref PubMed Scopus (53) Google Scholar, 9Wilks H.M. Hart K.W. Feeney R. Dunn C.R. Muirhead H. Chia W.N. Barstow D.A. Atkinson T. Clarke A.R. Holbrook J.J. Science. 1988; 242: 1541-1544Crossref PubMed Scopus (248) Google Scholar, 10Benkovic S.J. Fierke C.A. Naylor A.M. Science. 1988; 239: 1105-1110Crossref PubMed Scopus (135) Google Scholar) has not found GS mutants but only TS and DB mutants. Obviously, our conclusions are not final, and the reader is clearly encouraged to use any mutation experiment. In view of the above points, it seems to us that GS destabilization mechanisms cannot provide a general way of reducing Δg cat‡. Thus, one must look for ways by which enzymes stabilize the TS more than the GS. Finding such ways is far from trivial and requires one to address thetotal effect of the enzyme, while comparing it to the proper reference state in solution. Available experimental results do not yet tell us in a unique way how the total effect of the enzyme is distributed and what are the exact contributions of different free energy factors (e.g. strain, electrostatic, entropy, etc.). In particular, mutation experiments can help enormously by telling us what is the effect of different residues but cannot determine the origin of the overall catalysis. For example, mutating a group that forms a hydrogen bond (HB) to a negatively charged transition state in an active site, which is otherwise completely non-polar, will lead to a very large reduction in k cat. Some people upon being informed about this mutational effect will conclude that the native enzyme is a very good catalyst because its HB is so effective. However, a charge is very unstable in a non-polar environment, and a charged TS that is stabilized by a single HB will still leave us with an enzyme that destabilizes its TS relative to water (11Warshel A. Papazyan A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13665-13670Crossref PubMed Scopus (183) Google Scholar). Many reasonable proposals have been put forward to account for the catalytic power of enzymes. However, well defined energy considerations and computer modeling studies are now indicating that most of these proposals cannot account for large catalytic effects. This does not imply that such proposals have not been reasonable but rather that it is difficult to validate them without qualitative and sometimes quantitative energy considerations. To illustrate the effectiveness of energy considerations and computer modeling we consider below some of the main catalytic proposals and refer the reader to a more detailed analysis in Ref. 12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar. One of the most popular proposals is that enzymes work by providing a non-polar (or sometimes gas phase-like) environment that destabilizes highly charged ground states (13Crosby J. Stone R. Lienhard G.E. J. Am. Chem. Soc. 1970; 92: 2891-2900Crossref PubMed Scopus (177) Google Scholar, 14Dewar M.J. Dieter K.M. Biochemistry. 1988; 27: 3302-3308Crossref PubMed Scopus (31) Google Scholar, 15Dewar M.J.S. Storch D.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2225-2229Crossref PubMed Scopus (199) Google Scholar, 16Lee J.K. Houk K.N. Science. 1997; 276: 942-945Crossref PubMed Scopus (162) Google Scholar, 17Lightstone F.C. Zheng Y.-J. Maulitz A.H. Bruice T.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8417-8420Crossref PubMed Scopus (57) Google Scholar). As shown in Ref. 18Warshel A. Åqvist J. Creighton S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5820-5824Crossref PubMed Scopus (136) Google Scholar these proposals involve improper thermodynamic cycles and do not use a proper reference state. This amounts to ignoring the desolvation energy associated with taking the GS from water to a hypothetical non-polar enzyme site. With a proper reference state, one finds (18Warshel A. Åqvist J. Creighton S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5820-5824Crossref PubMed Scopus (136) Google Scholar) that a polar TS is less stable in non-polar sites than in water and that the GS destabilization does not help in increasing k cat/K m. In fact, many desolvation models (e.g. Refs. 13Crosby J. Stone R. Lienhard G.E. J. Am. Chem. Soc. 1970; 92: 2891-2900Crossref PubMed Scopus (177) Google Scholar, 16Lee J.K. Houk K.N. Science. 1997; 276: 942-945Crossref PubMed Scopus (162) Google Scholar, and 17Lightstone F.C. Zheng Y.-J. Maulitz A.H. Bruice T.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8417-8420Crossref PubMed Scopus (57) Google Scholar) involve ionized residues that will simply be unionized in non-polar sites. Moreover, in any specific case when the structure of the active site is known (e.g. Ref. 19Arjunan P. Umland T. Dyda F. Swaminathan S. Furey W. Sax M. Farrenkopf B. Gao Y. Zhang D. Jordan F. J. Mol. Biol. 1996; 256: 590-600Crossref PubMed Scopus (193) Google Scholar), one finds by current electrostatic models a very polar (rather than non-polar) active site environment near the chemically active part of the substrate. This idea (4Jencks W.P. Catalysis in Chemistry and Enzymology. Dover Publications, New York1986Google Scholar, 20Bruice T.C. Annu. Rev. Biochem. 1976; 45: 331-373Crossref PubMed Scopus (111) Google Scholar) implies that the reduction of Δg cat‡ is because of a restriction of motion of the reacting fragments of the substrate in the enzyme active site. This proposal involves ground state destabilization and is probably not so useful for enzymes that evolved by optimizing k cat/K m (see “The Real Problem is the Origin of the Reduction of Δg cat‡”). The interpretation of experiments in model compounds that were brought to support this proposal (4Jencks W.P. Catalysis in Chemistry and Enzymology. Dover Publications, New York1986Google Scholar, 20Bruice T.C. Annu. Rev. Biochem. 1976; 45: 331-373Crossref PubMed Scopus (111) Google Scholar) is now questioned by computational studies (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, 21Lightstone F.C. Bruice T.C. J. Am. Chem. Soc. 1996; 118: 2595-2605Crossref Scopus (185) Google Scholar), and it is also argued to be somewhat irrelevant (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar). It is still possible that entropic effects contribute a few kcal/mol to the reduction of Δg cat,‡, but a quantitative judgment of the importance of entropic effects will have to involve accurate simulations of this effect. This proposal (22Storm D.R. Koshland D.E. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 445-457Crossref PubMed Google Scholar) postulated a very narrow dependence of the transition state energy on the angle of approach of the reacting molecules. The corresponding rate acceleration is in conflict with the following. First, as was pointed out eloquently by Bruice and co-workers (23Bruice T.C. Brown A. Harris D.O. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 658-661Crossref PubMed Google Scholar) and confirmed by subsequent calculations (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, 25Dorigo A.E. Houk K.N. Liotta D. Advances in Molecular Modeling. JAI Press Inc., Greenwich, CT1988: 135-187Google Scholar), this proposal requires an unreasonably large force constant. Second, and in some respects more importantly, the proposal has not been properly defined, because it did not consider the corresponding effect of the reference reaction in water (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar). With a proper reference state, one finds that the proposed catalytic effect disappears unless the enzyme can constrain the reacting molecules in the ground state to the same narrow angular range as the corresponding range in the transition state of the solution reaction (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar). Such a situation is extremely unlikely in flexible enzymes and has little to do with orbitals because we have the same reacting orbitals in the protein and in solution. In this way we simply have another proposal of ground state destabilization by orientational entropy. Finally, a recent work (24Mesecar A.D. Stoddard B.L. Koshland Jr., D.E. Science. 1997; 277: 202-206Crossref PubMed Scopus (202) Google Scholar) that supported the orbital steering model can be interpreted differently. 3Glennon, T. M., and Warshel, A. (1998) J. Am. Chem. Soc., in press. This proposal (26Frey P.A. Whitt S.A. Tobin J.B. Science. 1994; 264: 1927-1930Crossref PubMed Scopus (731) Google Scholar, 27Cleland W.W. Kreevoy M.M. Science. 1994; 264: 1887-1890Crossref PubMed Scopus (1048) Google Scholar, 28Gerlt J.A. Gassman P.G. Biochemistry. 1993; 32: 11943-11952Crossref PubMed Scopus (369) Google Scholar) involves three points: (a) ionic HBs that include a negatively charged proton acceptor contribute in a major way to transition state stabilization; (b) the catalytic effect of HBs is associated with their preorganization (28Gerlt J.A. Gassman P.G. Biochemistry. 1993; 32: 11943-11952Crossref PubMed Scopus (369) Google Scholar); and (c) hydrogen bonds between the enzyme and charged transition states of substrates involve a much larger covalent character (X −δ···H···Y−δ) than the corresponding hydrogen bonds in solution; otherwise we have a regular (X − H–Y) electrostatic HB. The first two parts of the proposal are correct but were introduced long ago as a major part of the electrostatic effect of enzymes (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, 29Wilkinson A.J. Fersht A.R. Blow D.M. Winter G. Biochemistry. 1983; 22: 3581-3586Crossref PubMed Scopus (256) Google Scholar, 30Warshel A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5250-5254Crossref PubMed Scopus (437) Google Scholar). Our analysis indicates that the new assumption of a larger covalent character for HBs in enzymes than in solution results in a reduction of the catalytic effect of these HBs (11Warshel A. Papazyan A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13665-13670Crossref PubMed Scopus (183) Google Scholar, 31Warshel A. Papazyan A. Kollman P.A. Science. 1995; 269: 102-106Crossref PubMed Scopus (328) Google Scholar). The reason is that LBHBs can be effective only in a non-polar environment, but such an environment results in HBs that give less TS stabilization than regular electrostatic HBs. That is, LBHBs can provide very large stabilization to charged TSs in a non-polar enzyme surrounded by non-polar solvent rather than water. However, immersing this enzyme in water will create a very poor catalyst because of the above mentioned desolvation problem. In a polar environment, one finds (11Warshel A. Papazyan A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13665-13670Crossref PubMed Scopus (183) Google Scholar) that an LBHB provides less TS stabilization than a regular HB. In this respect, it is crucial to realize that the alternative to an ionic LBHB is an ionic HB, which acts through electrostatic effects. Thus, in a complete contrast to some assertions (32Pan Y. McAllister M.A. J. Am. Chem. Soc. 1998; 120: 166-169Crossref Scopus (83) Google Scholar) we never suggested that the alternative to an ionic LBHB is a neutral (X H–Y) HB. All discussions of strong HBs in enzymes involved ionic TSs, and it was never suggested that regular catalytic HBs are weak (33Cassidy C.S. Lin J. Frey P.A. Biochemistry. 1997; 36: 4576-4584Crossref PubMed Scopus (173) Google Scholar) but demonstrated that HBs are strong (34Warshel A. Sussman F. Hwang J.-K. J. Mol. Biol. 1988; 201: 139-159Crossref PubMed Scopus (172) Google Scholar). Distinguishing between LBHBs and HBs is a question of interpretation of experiments rather than experiments alone, and current experiments cannot be interpreted unambiguously (11Warshel A. Papazyan A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13665-13670Crossref PubMed Scopus (183) Google Scholar). A case in point is a recent NMR study (35Ash E.L. Sudmeier J.L. DeFabo E.C. Bachovchin W.W. Science. 1997; 278: 1128-1132Crossref PubMed Scopus (177) Google Scholar) that suggested a very different interpretation from that of previous studies (26Frey P.A. Whitt S.A. Tobin J.B. Science. 1994; 264: 1927-1930Crossref PubMed Scopus (731) Google Scholar). Energy considerations can help in reaching a more unique analysis of the nature of ionic HBs in enzyme (31Warshel A. Papazyan A. Kollman P.A. Science. 1995; 269: 102-106Crossref PubMed Scopus (328) Google Scholar). For example, in the case of serine proteases, it was shown (11Warshel A. Papazyan A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13665-13670Crossref PubMed Scopus (183) Google Scholar, 31Warshel A. Papazyan A. Kollman P.A. Science. 1995; 269: 102-106Crossref PubMed Scopus (328) Google Scholar) that the pK a of the catalytic His-57 and Asp-102 must be very different to stabilize the transition state (relative to water), and thus we cannot have an LBHB in this system. The same arguments can be used to show that a new analysis (33Cassidy C.S. Lin J. Frey P.A. Biochemistry. 1997; 36: 4576-4584Crossref PubMed Scopus (173) Google Scholar, 36Cleland W.W. Frey P.A. Gerlt J.A. J. Biol. Chem. 1998; 273: 25529-25532Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar) of a negatively charged TS analog of serine proteases proves that the LBHB hypothesis is very problematic. That is, in this case, the pK a of His-57 is raised to about 12, and this was attributed to binding-induced ground state strain, which has been assumed to be consistent with the LBHB hypothesis (33Cassidy C.S. Lin J. Frey P.A. Biochemistry. 1997; 36: 4576-4584Crossref PubMed Scopus (173) Google Scholar). However, the presence of a negatively charged TS analog stabilizes the protonated form of His-57 much more than it destabilized the more distant and negatively charged Asp-102. Thus, the TS analog increases rather than decreases the pK a difference in the Asp-His pair, which is, of course, consistent with the experimentally observed pK a. This trend contradicts the LBHB proposal, which requires a matching pK a. Furthermore, the proposed strain effect is inconsistent with mutational studies that moved the COO− group from residue 102 to residue 214 and still obtained significant catalysis (37Corey D.R. McGrath M.E. Vásquez J.R. Fletterick R.J. Craik C.S. J. Am. Chem. Soc. 1992; 114: 4905-4907Crossref Scopus (31) Google Scholar). It is useful to realize that recent theoretical studies (32Pan Y. McAllister M.A. J. Am. Chem. Soc. 1998; 120: 166-169Crossref Scopus (83) Google Scholar, 38Pan Y. McAllister M.A. J. Org. Chem. 1997; 62: 8171-8176Crossref PubMed Scopus (40) Google Scholar), which were discussed in the first minireview in this series (36Cleland W.W. Frey P.A. Gerlt J.A. J. Biol. Chem. 1998; 273: 25529-25532Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar), in support of the LBHB hypothesis reflect an improper analysis of an irrelevant system (i.e. no protein is studied) and apparently fail to address the relevant question (i.e. confuse ionic HB with LBHB). That is, the theoretical study (38Pan Y. McAllister M.A. J. Org. Chem. 1997; 62: 8171-8176Crossref PubMed Scopus (40) Google Scholar) that is taken as a support of the LBHB hypothesis (36Cleland W.W. Frey P.A. Gerlt J.A. J. Biol. Chem. 1998; 273: 25529-25532Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar) attempted to model an enzyme by placing two water molecules around an HB in the gas phase. The resulting ionic HB was found to be extremely strong relative to the improperreference of the dissociated HB in the gas phase. Unfortunately, this strong HB has a very high energy (relative to its energy in water), because it involves a charge in a non-polar environment. Thus, we have here another illustration of the anticatalytic nature of LBHBs. Similarly, the computational studies of an LBHB in polar solvent (32Pan Y. McAllister M.A. J. Am. Chem. Soc. 1998; 120: 166-169Crossref Scopus (83) Google Scholar) that were brought in support of the LBHB proposal (36Cleland W.W. Frey P.A. Gerlt J.A. J. Biol. Chem. 1998; 273: 25529-25532Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar) involved a comparison of an ionic LBHB and a neutral HB. This has little to do with the LBHB issue which involves the comparison of an ionic LBHB and an ionic HB (see above). Furthermore, Ref. 32Pan Y. McAllister M.A. J. Am. Chem. Soc. 1998; 120: 166-169Crossref Scopus (83) Google Scholar grossly overestimates the actual strength (39Guthrie J.P. Chem. Biol. 1996; 3: 163-170Abstract Full Text PDF PubMed Google Scholar) of ionic HBs in polar solvents. This proposal (40Careri G. Fasella P. Gratton E. Annu. Rev. Biophys. Bioeng. 1979; 8: 69-97Crossref PubMed Scopus (180) Google Scholar, 41Gavish B. Werber M.M. Biochemistry. 1979; 18: 1269-1275Crossref PubMed Scopus (193) Google Scholar, 42McCammon J.A. Wolynes P.G. Karplus M. Biochemistry. 1979; 18: 927-942Crossref PubMed Scopus (280) Google Scholar) implies that the enzyme might induce special fluctuations that do not obey the Eyring's absolute rate theory, leading to a transmission factor that is much smaller than unity (note that the transmission factor contains all dynamic effects (see Ref. 43Warshel A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 444-448Crossref PubMed Scopus (157) Google Scholar) and that factors such as activation energies that can be obtained by Monte Carlo simulations, rather than only by Molecular Dynamics simulations, are not dynamic effects). However, our simulation studies established quite early that the reactive fluctuations are similar in enzymes and solutions (43Warshel A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 444-448Crossref PubMed Scopus (157) Google Scholar) and that the transmission factor is similar in both cases (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, 34Warshel A. Sussman F. Hwang J.-K. J. Mol. Biol. 1988; 201: 139-159Crossref PubMed Scopus (172) Google Scholar). The same conclusion was recently reached by others (44Neria E. Karplus M. Chem. Phys. Lett. 1997; 267: 23-30Crossref Scopus (97) Google Scholar). Nevertheless, it was implied in Ref. 44Neria E. Karplus M. Chem. Phys. Lett. 1997; 267: 23-30Crossref Scopus (97) Google Scholar that the non-equilibrium solvation in enzymes is fundamentally different from the corresponding effects in solution. However, this conclusion was reached without attempting to calculate the dynamics in a solvent cage or to evaluate the activation free energy and its non-equilibrium contributions in the enzyme and in solution. In fact, early studies that compared the dynamics of enzymatic reactions to the corresponding reaction in solution found that both systems have similar solvent fluctuations and similar solute fluctuations (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, 43Warshel A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 444-448Crossref PubMed Scopus (157) Google Scholar). The only difference is that the amplitude of the fluctuations is smaller in the more preorganized environment. Interestingly, a recent simulation study 4A. Warshel and J. Bentzien, submitted for publication. established that non-equilibrium solvation effects are of similar magnitude in enzymes and in the corresponding solution reaction. This proposal (45Cha Y. Murray C.J. Klinman J.P. Science. 1989; 243: 1325-1330Crossref PubMed Scopus (446) Google Scholar, 46Bahnson B.J. Colby T.D. Chin K.J. Goldstein B.M. Klinman J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12797-12802Crossref PubMed Scopus (178) Google Scholar) suggests that enzymes can accelerate their reactions by exploiting nuclear tunneling effects. Well chosen experimental studies have indicated that the degree of tunneling changes when the rate constant changes upon mutations (46Bahnson B.J. Colby T.D. Chin K.J. Goldstein B.M. Klinman J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12797-12802Crossref PubMed Scopus (178) Google Scholar). However, it was not yet demonstrated that the change in rate constants is because of tunneling (because the large effect of mutations did not change upon isotopic substitution). It is quite possible, for example, that the mutations change the reorganization energy, and this changes the tunneling rather than the other way around. Simulation studies (e.g. Ref. 47Hwang J.-K. Warshel A. J. Am. Chem. Soc. 1996; 118: 11745-11751Crossref Scopus (193) Google Scholar) have shown that enzymatic reactions may involve significant tunneling effects, but the corresponding catalytic effects are not expected to be large because similar tunneling corrections occur in the enzyme and the reference solution reaction. In summary, non-negligible contributions may still be provided by entropy, tunneling, and perhaps some LBHB character, although these are not supported by current simulation studies. Large contributions are expected from electrostatic effects (see below). As indicated above it is frequently simpler to show what does not lead to catalysis than to establish what does. Even the interpretation of mutation experiments is far from being unique. For example, the large observed effect of mutating Asp-32 in subtilisin can be interpreted as an electrostatic effect (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, 48Warshel A. Naray-Szabo G. Sussman F. Hwang J.-K. Biochemistry. 1989; 28: 3629-3637Crossref PubMed Scopus (411) Google Scholar), an LBHB effect (26Frey P.A. Whitt S.A. Tobin J.B. Science. 1994; 264: 1927-1930Crossref PubMed Scopus (731) Google Scholar,27Cleland W.W. Kreevoy M.M. Science. 1994; 264: 1887-1890Crossref PubMed Scopus (1048) Google Scholar), and an entropic effect (49Craik C.S. Roczniak S. Largman C. Rutter W.J. Science. 1987; 237: 909-913Crossref PubMed Scopus (286) Google Scholar). It seems to us that only a quantitative, structurally based analysis can help in providing a more unique interpretation. The reason is quite simple; we are dealing with a very complex system with many free energy contributions, and a proper analysis requires a quantitative model that can handle such complexity and predict rather than assume the relevant contributions. Such an analysis can be done by computer simulations. Computer-aided methods for correlating the structures of enzymes have been developed (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar), and some of these approaches, which are referred to as hybrid quantum mechanics/molecular mechanics (QM/MM) methods (50Warshel A. Levitt M. J. Mol. Biol. 1976; 103: 227-249Crossref PubMed Scopus (3695) Google Scholar), are becoming very popular (e.g. Refs. 51Bash P.A. Field M.J. Davenport R.C. Petsko G.A. Ringe D. Karplus M. Biochemistry. 1991; 30: 5826-5832Crossref PubMed Scopus (260) Google Scholar and 52Mulholland A.J. Grant G.H. Richards W.G. Protein Eng. 1993; 6: 133-147Crossref PubMed Scopus (68) Google Scholar). Yet at present, the only approach that reaches the level of quantitative analysis is the empirical valence bond (EVB) method (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar,53Åqvist J. Warshel A. Chem. Rev. 1993; 93: 2523-2544Crossref Scopus (760) Google Scholar). This approach is quantitative because it does not attempt to calculate the energetics of bond-making/bond-breaking processes by anab initio first principle approach (which requires at present too much computer power) but focuses only on thechange in such energetics upon moving from the reference solvent cage to the enzyme active site. This is done by parameterizing the EVB Hamiltonian on ab initio studies and on experimental information about chemical reactions in solution and then keeping the parameters completely unchanged when exploring the free energy of moving from solution to enzymes. Thus, we can determine quantitatively free energy contributions to catalysis without the use of any free parameters. The EVB model has been recently adopted by other research groups who found its features useful for studying reactions in solutions and enzymes (44Neria E. Karplus M. Chem. Phys. Lett. 1997; 267: 23-30Crossref Scopus (97) Google Scholar, 54Lobaugh J. Voth G.A. J. Chem. Phys. 1996; 104: 2056-2069Crossref Scopus (331) Google Scholar, 55Kim H.J. Hynes J.T. J. Am. Chem. Soc. 1992; 114: 10508-10537Crossref Scopus (130) Google Scholar, 56Bala P. Grochowski P. Lesyng B. McCammon J.A. J. Phys. Chem. 1996; 100: 2535-2545Crossref Scopus (91) Google Scholar). All EVB studies (e.g. Refs. 12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, 34Warshel A. Sussman F. Hwang J.-K. J. Mol. Biol. 1988; 201: 139-159Crossref PubMed Scopus (172) Google Scholar, 48Warshel A. Naray-Szabo G. Sussman F. Hwang J.-K. Biochemistry. 1989; 28: 3629-3637Crossref PubMed Scopus (411) Google Scholar, 53Åqvist J. Warshel A. Chem. Rev. 1993; 93: 2523-2544Crossref Scopus (760) Google Scholar, and 57Fuxreiter M. Warshel A. J. Am. Chem. Soc. 1998; 120: 183-194Crossref Scopus (102) Google Scholar) and even more qualitative QM/MM studies (e.g. Refs. 51Bash P.A. Field M.J. Davenport R.C. Petsko G.A. Ringe D. Karplus M. Biochemistry. 1991; 30: 5826-5832Crossref PubMed Scopus (260) Google Scholar and 52Mulholland A.J. Grant G.H. Richards W.G. Protein Eng. 1993; 6: 133-147Crossref PubMed Scopus (68) Google Scholar), studies that took the protein plus solvent into account, have indicated repeatedly that enzyme catalysis is due mainly to electrostatic effects. It is important to note here that using computational approaches does not always guarantee relevant results. As is now becoming clear in the computational community, high level ab initio calculations that do not include the enzyme cannot tell us much about the role of the enzyme. Some readers might assume that the importance of electrostatic effects in enzyme catalysis is a rather trivial idea that has been established in the early days of the field. However, although electrostatic effects were proposed quite early (58Vernon C.A. Proc. R. Soc. Lond. B Biol. Sci. 1967; 167: 389Crossref PubMed Google Scholar), they were found to be inconsistent with all early experimental studies. That is, experiments with model compounds in solutions show very small electrostatic effects (20Bruice T.C. Annu. Rev. Biochem. 1976; 45: 331-373Crossref PubMed Scopus (111) Google Scholar, 59Fife T.H. Jaffe S.H. Natarajan R. J. Am. Chem. Soc. 1991; 113: 7646-7653Crossref Scopus (13) Google Scholar). Similarly, changes of ionic strength that were used to probe electrostatic effects did not produce major changes in enzyme activity. Thus, until the emergence of genetic engineering, there was no major experimental evidence for large electrostatic contributions to catalytic effects of enzymes. Theoretical studies, on the other hand, pointed to such effects repeatedly (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, 30Warshel A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5250-5254Crossref PubMed Scopus (437) Google Scholar, 50Warshel A. Levitt M. J. Mol. Biol. 1976; 103: 227-249Crossref PubMed Scopus (3695) Google Scholar). The difficulties in early experimental verification were traced to the fact that the interior of enzymes cannot be probed by external perturbation and that experiments with model compounds in solution have not produced large electrostatic effects because of large dielectric effects. The proposal that enzymes work by electrostatic stabilization mechanisms has to overcome one major fundamental problem. That is, it is not obvious how an enzyme active site can provide more electrostatic stabilization than water. More specifically, computer simulation studies (34Warshel A. Sussman F. Hwang J.-K. J. Mol. Biol. 1988; 201: 139-159Crossref PubMed Scopus (172) Google Scholar) indicated that the actual electrostatic interaction between the enzyme and the TS of its substrate, ΔG Qμ, is similar to that between water and the corresponding TS. If the interactions are similar, one would expect no catalytic effect. The solution to this fundamental problem has been provided in an early work (30Warshel A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5250-5254Crossref PubMed Scopus (437) Google Scholar). This work pointed out that in polar solvents about half of the energy gained from charge-dipole interaction is spent on changing the dipole-dipole interaction, ΔG μμ, so that the free energy of solvation of the transition state is given by (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar) the following equation.ΔGsol≅ΔGQμ+ΔGμμ≅ΔGQμ−1/2ΔGQμ=1/2ΔGQμ(Eq. 1) In proteins, however, the active site dipoles associated with polar groups, internal water molecules, and ionized residues are already partially oriented toward the transition state charge center. Thus, ΔG μμ is smaller than in water, and less free energy is spent on creating the oriented dipoles of the protein transition state (Fig. 3). The free energy term ΔG μμ is basically the so-called “reorganization energy” (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, 60Marcus R.A. J. Chem. Phys. 1956; 24: 966-978Crossref Scopus (5260) Google Scholar) for the process of forming the transition state charges. For example, in water we have to break water-water interactions to form good hydrogen bonds to the TS. In the enzyme, on the other hand, the hydrogen bonds are already partially oriented toward the transition state charges (34Warshel A. Sussman F. Hwang J.-K. J. Mol. Biol. 1988; 201: 139-159Crossref PubMed Scopus (172) Google Scholar). The prediction that the folding energy is used to preorient the enzyme dipoles is supported by the finding that mutations that increase Δg cat‡ also increase the folding energy (61Shoichet B.K. Baase W.A. Kuroki R. Matthews B.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 452-456Crossref PubMed Scopus (585) Google Scholar). The idea that enzymes use preorganized dipoles to catalyze their reactions should not be confused with the role of the Marcus reorganization energy in enzymatic reactions. That is, the activation energy for chemical reactions can be written as (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, 62Schweins T. Warshel A. Biochemistry. 1996; 35: 14232-14243Crossref PubMed Scopus (82) Google Scholar) follows,Δg‡=(ΔG0+λ)2/4λ−H12+H122/(λ+ΔG0)(Eq. 2) where ΔG 0 is the free energy of moving from the reactant to the product (or an intermediate) and λ is the so-called “solvent reorganization energy,” which mainly reflects the changes in the solvent-solvent interaction during the reaction.H 12 describes the mixing between the reactant and product state. The first term in this equation is the well known Marcus expression for electron transfer reactions, whereas the other terms allow the treatment of regular chemical reactions. At any rate, one can use the first term in Equation 2 to correlate small changes of Δg ‡ with the effect of mutations. Consideration of the Marcus term led different workers to argue that Δg ‡ can be reduced by reducing ΔG 0 (63Albery W.J. Knowles J.R. Biochemistry. 1976; 15: 5631-5640Crossref PubMed Scopus (592) Google Scholar) or by reducing λ (64Gerlt J.A. Gassman P.G. J. Am. Chem. Soc. 1993; 115: 11552-11568Crossref Scopus (383) Google Scholar). Unfortunately, this argument does not help in elucidating the origin of enzyme catalysis; instead of stating that Δg cat‡ is reduced, we state now that ΔG 0 and/or λ are reduced. However, we are still left without explaining how these free energies are reduced. Furthermore, insightful but incorrect suggestions that λ is reduced by having non-polar active sites (65Krishtalik L.I. J. Theor. Biol. 1985; 112: 251-264Crossref PubMed Scopus (23) Google Scholar) does not resolve this problem; although non-polar environment does reduce λ, it would lead to an increase of ΔG 0 and to anticatalytic effects (see discussion in Ref. 66Yadav A. Jackson R.M. Holbrook J.J. Warshel A. J. Am. Chem. Soc. 1991; 113: 4800-4805Crossref Scopus (110) Google Scholar). What is missing in this proposal is the idea that enzymes reduce both λ and ΔG 0 by preorganized polar (rather than non-polar) environment as established by simulation studies (12Warshel A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, 53Åqvist J. Warshel A. Chem. Rev. 1993; 93: 2523-2544Crossref Scopus (760) Google Scholar, 66Yadav A. Jackson R.M. Holbrook J.J. Warshel A. J. Am. Chem. Soc. 1991; 113: 4800-4805Crossref Scopus (110) Google Scholar). The origin of this reduction is directly connected to the above mentioned preorganized polar environment. Such an environment stabilizes charged intermediates by not investing in ΔG μμ, and as a result of having preorganized dipoles it also reduces λ. If enzymes really use their preoriented environment to stabilize the transition state, then we understand why it was so difficult to elucidate the origin of enzyme catalysis. In this case, the catalytic energy is not stored in the enzyme-substrate interaction but in the enzyme itself. Thus, for example, the reduction of Δg cat‡ is not associated with the entropy loss upon assembly of the substrate fragments but with the free energy invested in fixing the environment. In view of the length of our arguments, we will summarize their main points as follows. (i) Enzymes attain a largek cat by providing more stabilization to the charges of transition states than the corresponding stabilization in water. (ii) It is not true that evolution can increasek cat by using any free energy factor, because many factors do not provide a physical way of doing so. (iii) Although binding of the “non-chemical part” of the substrate can help in stabilizing the TS, it cannot help in providing a large reduction to Δg cat‡. Such a reduction requires an active site that is able to stabilize the reacting (chemical) part of the TS more than the corresponding solvent cage does. (iv) The reduction of Δg cat‡ is accomplished by electrostatic stabilization, which is due to a preorganized polar environment (30Warshel A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5250-5254Crossref PubMed Scopus (437) Google Scholar).