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

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. Vell