Electrostatic Origin of the Catalytic Power of Enzymes and the Role of Preorganized Active Sites
Year: 1998
Type: review
Abstract: 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, Biochemistry. 1988; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1985; 82: PubMed Scopus Google Scholar, Science. PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). As shown in Ref. A. J. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google these proposals involve and do not use a proper reference This to the desolvation energy associated with the GS from water to a non-polar enzyme a proper reference one A. J. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) that a TS is in non-polar than in water and that the GS destabilization does not help in increasing k cat/K m. In many desolvation (e.g. J. Stone R. Lienhard G.E. J. Am. Chem. Soc. 1970; 92: 2891-2900Crossref PubMed Scopus (177) Google Scholar, Science. PubMed Scopus Google Scholar, and Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) involve residues that will be in non-polar in any case when the of the active is known (e.g. Ref. P. T. S. W. J. 1996; PubMed Scopus Google Scholar), one by a very than active environment the active of the This (4Jencks W.P. Catalysis in Chemistry and Enzymology. Dover Publications, New York1986Google Scholar, PubMed Scopus (111) Google Scholar) that the reduction of Δg cat‡ is because of a of of the of the substrate in the enzyme active This ground state destabilization and is not so for enzymes that evolved by k cat/K m (see is the of the of Δg The of experiments in that were to this (4Jencks W.P. Catalysis in Chemistry and Enzymology. Dover Publications, New York1986Google Scholar, PubMed Scopus (111) Google Scholar) is now by computational studies A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, J. Am. Chem. Soc. 1996; Scopus Google Scholar), and it is also to be A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar). It is still that effects a to the reduction of Δg but a quantitative of the of effects will have to involve of this This Proc. Natl. Acad. Sci. U. S. A. 1970; PubMed Google Scholar) a very of the transition state energy the of of the The rate is in with the as was out eloquently by and co-workers A. Proc. Natl. Acad. Sci. U. S. A. PubMed Google Scholar) and by A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, in Inc., Scholar), this requires an large and in some more the has not been because it not consider the effect of the reference in water A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar). a proper reference one that the catalytic effect the enzyme can the in the ground state to the same as the in the transition state of the A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar). a is in enzymes and has to do with because we have the same in the and in solution. 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PubMed Scopus Google and Eng. 1993; 6: PubMed Scopus Google Scholar), studies that the into have indicated that enzyme is due to effects. It is to here that computational does not As is now clear in the computational that do not the enzyme cannot tell us much about the of the Some that the of effects in enzyme is a rather trivial that has been in the of the However, effects were quite C.A. Proc. R. Soc. Sci. PubMed Google Scholar), were found to be with experimental experiments with in very effects PubMed Scopus (111) Google Scholar, R. J. Am. Chem. Soc. Scopus Google Scholar). Similarly, changes of that were used to effects not major changes in enzyme Thus, the of there was major experimental for large contributions to catalytic effects of enzymes. the other to such effects A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. J. PubMed Scopus Google Scholar). The in experimental were to the fact that the of enzymes cannot be by and that experiments with in have not large effects because of large effects. The that enzymes work by stabilization mechanisms has to one major fundamental it is not obvious how an enzyme active can provide more stabilization than computer studies A. J. 1988; PubMed Scopus Google Scholar) indicated that the actual between the enzyme and the TS of its is to that between water and the If the are one would catalytic The to this fundamental has been provided in an work A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). This work out that in about of the energy from is changing the so that the free energy of of the transition state is by A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar) the In the active associated with water and residues are the transition state charge Thus, is than in and free energy is the of the transition state The free energy is basically the A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, J. Chem. 24: Scopus Google Scholar) for the of the transition state For example, in water we have to to good hydrogen to the In the enzyme, the other the hydrogen are the transition state A. J. 1988; PubMed Scopus Google Scholar). The that the energy is used to the enzyme is by the finding that mutations that increase Δg cat‡ also increase the energy R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). The that enzymes use to their reactions should not be with the of the energy in the energy for reactions can be as A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, T. A. Biochemistry. 1996; PubMed Scopus Google Scholar) is the free energy of from the to the (or an and is the which the changes in the the the between the and The in this is the well known for the other allow the of any one can use the in to changes of Δg ‡ with the effect of of the different workers to that Δg ‡ can be reduced by reducing Biochemistry. PubMed Scopus Google Scholar) by reducing J.A. J. Am. Chem. Soc. 1993; Scopus Google Scholar). this does not help in the origin of enzyme of that Δg cat‡ is we state now that are However, we are still without how these free are but that is reduced by non-polar active J. 1985; PubMed Scopus Google Scholar) does not this non-polar environment does reduce it would lead to an increase of and to effects (see in Ref. A. Holbrook J.J. A. J. Am. Chem. Soc. Scopus Google Scholar). is in this is the that enzymes reduce and by than environment as by studies A. Computer Modeling of Chemical Reactions in Enzymes and Solutions. John Wiley & Sons, Inc., New York1991Google Scholar, J. A. Chem. 1993; 93: Scopus Google Scholar, A. Holbrook J.J. A. J. Am. Chem. Soc. Scopus Google Scholar). The origin of this reduction is to the above an environment charged by not in and as a of it also If enzymes use their environment to stabilize the transition then we it was so difficult to the origin of enzyme catalysis. In this the catalytic energy is not in the but in the enzyme Thus, for example, the reduction of Δg cat‡ is not associated with the upon of the substrate but with the free energy in the In view of the of our we will their main as Enzymes a cat by providing more stabilization to the of transition states than the stabilization in It is not true that evolution can cat by any free energy because many factors do not provide a way of binding of the of the substrate can help in the TS, it cannot help in providing a large reduction to Δg cat‡. a reduction requires an active that is to stabilize the of the TS more than the The reduction of Δg cat‡ is accomplished by stabilization, which is due to a environment A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar).
Source: Journal of Biological Chemistry
Author: Arieh Warshel
Institution: University of Southern California
Language: English
Subfield: Organic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W2107025108