Year: 1998
Type: review
Abstract: transition state analog. The question of how enzymes greatly enhance the rate of reactions has been discussed for years but remains a vigorously debated issue. Rapid progress has been made on the mechanism of individual enzymes by a combination of kinetic, chemical, and structural approaches. The push and pull of electrons and the resulting bond changes are well understood for many enzymes. However, the larger question of general features that enzymes use to produce rate accelerations of 108–1015 has remained a contentious issue. We believe that such rate accelerations can be readily explained by reasonable physical principles (1Cannon W.R. Singleton S.F. Benkovic S.J. Nat. Struct. Biol. 1996; 3: 821-833Crossref PubMed Scopus (134) Google Scholar). Though much of this understanding stems from research done in the 1970s and 1980s, these insights are often underappreciated or even completely neglected when examining enzymic rate accelerations. Perhaps this is because of our intense focus on enzymes themselves and relative neglect of the reference solution reactions to which they are compared, consequentially, resulting in a mechanistic tunnel vision. Drawing on an earlier analysis (1Cannon W.R. Singleton S.F. Benkovic S.J. Nat. Struct. Biol. 1996; 3: 821-833Crossref PubMed Scopus (134) Google Scholar) and work cited herein, we wish to examine a popular model for comparing the rates of catalyzed and uncatalyzed reactions, that is, the thermodynamic cycle as set up in Scheme 1, which compares a transformation of a reactant (S) in solution through its transition state (S‡) with the same reaction catalyzed by an enzyme (E) from its ground state (ES) to its transition state (ES‡). E+S →knon E+S‡K=1↑↑KTS E+S ←Km ES →kcat ES‡SCHEME IThe solution reaction is characterized by the reaction ratek non, and the enzyme-catalyzed reaction is characterized by the Michaelis-Menten constantsk cat and Km . An apparent equilibrium constant for the dissociation of the transition state of the reactant from the enzyme can be obtained using transition state theory (2Kurz J.L. J. Am. Chem. Soc. 1963; 85: 987-991Crossref Scopus (128) Google Scholar), K TS =((k cat /Km)/k non)−1. The value of K TS is generally significantly smaller than the dissociation constant for the substrate from the enzyme, KS, leading to a large and favorable free energy of binding for the enzyme and S‡ (3Wolfenden R. Acc. Chem. Res. 1972; 5: 10-18Crossref Scopus (546) Google Scholar, 4Lienhard G.E. Science. 1973; 180: 149-154Crossref PubMed Scopus (417) Google Scholar, 5Radzicka A. Wolfenden R. Science. 1995; 267: 90-93Crossref PubMed Scopus (741) Google Scholar). As a result of the small value of K TS, analogs of the reactant in its transition state geometry (transition state analog (TSA)1) have been sought as enzyme inhibitors. In addition, TSAs and the scenario of Scheme 1 have been used as the foundation for the design of catalysts from antibodies, in which immunization against a TSA is used to elicit the formation of antibodies that strongly bind the TSA. The thermodynamic cycle set up in Scheme 1 is useful as a model for understanding many aspects of rate accelerations. However, as in all models of complex phenomena, its limitations must also be kept in mind. Without conjecture, all we can say from the analysis of the thermodynamic cycle is that, if the virtual binding of the reactant in its transition state geometry were physically able to occur, the process would be very favorable in terms of free energy. The difference in the apparent free energy of binding of S‡ to the enzyme relative to S may result from two scenarios. In the first scenario, significantly stronger interactions may exist between the enzyme and the reactant in its transition state configuration than in the enzymic ground state configuration. In this case, as the enzymic reaction proceeds along the lower path in Scheme 1, the binding of the reactant to the enzyme increases significantly. As an example, it has been proposed that short, strong hydrogen bonds could account for much of the “missing” energy required for the differential stabilization of the ES‡ complex compared with the ground state ES complex (6Cleland W.W. Kreevoy M.M. Science. 1994; 264: 1887-1890Crossref PubMed Scopus (1048) Google Scholar) (see the first minireview in this series by Cleland et al. (48Cleland 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) for details). The second possibility is that the free energy of interaction between the solvent environment and S‡ is much less favorable than between the solvent and S. Then, as the solution reaction proceeds toward the transition state along the upper path in Scheme 1, the free energy change includes a large and significant contribution from solvation. Although it is clear that the correct answer is a combination of the two scenarios, it is important for catalyst design purposes to determine their relative contributions toward enzymic rate accelerations. In the limiting cases, the former perspective incorporates all of the catalytic prowess into the interactions that develop exclusively within the ES‡ complex, whereas the latter perspective regards the solvent as being a major determinant of the rate of the uncatalyzed reaction. In the latter, the majority of the work in lowering the free energy barrier to the reaction occurs as a result of the binding process and not unusually strong interactions between the enzyme and substrate in theES‡ complex (see Refs. 3Wolfenden R. Acc. Chem. Res. 1972; 5: 10-18Crossref Scopus (546) Google Scholar and 7Wolfenden R. Science. 1983; 222: 1087-1093Crossref PubMed Scopus (107) Google Scholar for earlier views on this subject). A useful exercise is to plot K TS againstk cat and k non for a series of reactions where the rates of both the enzymic reaction and an appropriate model solution reaction are known. If enzymes predominantly function by interacting with the transition state configuration of the reactant dramatically more strongly than with the ground state configuration of the reactant, then we might expect that the most “powerful” enzymes, as determined by the lowest values of K TS, would also be the enzymes that have the fastest turnover rates, k cat. In contrast, if large rate accelerations (smallK TS) are due in large part to retardation of the solution reaction by the solvent environment, then such enzymes would be associated with a very slow solution reaction. Fortunately, Radzicka and Wolfenden (5Radzicka A. Wolfenden R. Science. 1995; 267: 90-93Crossref PubMed Scopus (741) Google Scholar, 8Bearne S.L. Wolfenden R. J. Am. Chem. Soc. 1996; 117: 9588-9589Crossref Scopus (31) Google Scholar, 9Radzicka A. Wolfenden R. J. Am. Chem. Soc. 1996; 118: 6105-6109Crossref Scopus (403) Google Scholar) among others have studied such model solution reactions and have been able to acquire valuable data for comparison. Fig. 1 comparesK TS with both k cat and k non for a number of such reactions. Whereas the reaction rates for the solution reactions (k non) are highly correlated with K TS(r = 0.96), the enzymic turnover rates (k cat) are poorly correlated with eitherK TS (r = 0.50) or the observed rate enhancement (k cat /k non, data not shown). Moreover, the relationship between changes in K TS and k non is essentially one-to-one, whereas in comparisonk cat remains essentially unchanged asK TS decreases dramatically (or −logK TS increases in Fig. 1). Although the turnover rates for most enzymes are limited by release of the product from the enzyme and not by the chemical event, nevertheless it can be clearly seen that the kinetics of the reactions in solution are the dominant contribution to the range of K TS values. Thus, although KTS is generally taken as a measure of an enzyme's catalytic proficiency, it cannot be inferred thatKTS is a reflection of the interactions that develop within ES‡. The stability of biomolecules in solution and in the absence of a specific catalyst is essential for the existence of metabolic pathways. Are these solution reactions intrinsically slow or does the solvent environment act to retard the rate that would be observed in the absence of such an environment? Experimental (10Bruice T.C. Benkovic S.J J. Am. Chem. Soc. 1963; 85: 1-8Crossref Scopus (88) Google Scholar) and theoretical (11Page M.I. Jencks W.P. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 1678-1683Crossref PubMed Scopus (1128) Google Scholar) studies suggested that the rates of bimolecular reactions in solution are slow in large part because of the entropic penalty involved in bringing the two reactants together into a bimolecular complex; three translational and three rotational degrees of freedom are lost in the association reaction and are apparently uncompensated for by the six new internal rotations and vibrations of the bimolecular complex (10Bruice T.C. Benkovic S.J J. Am. Chem. Soc. 1963; 85: 1-8Crossref Scopus (88) Google Scholar,11Page M.I. Jencks W.P. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 1678-1683Crossref PubMed Scopus (1128) Google Scholar). However, the majority of reactions in Fig. 1 are either unimolecular decomposition reactions or pseudo-first order reactions. Although loss of intrinsic entropy during an association process may be an important aspect of enzyme catalysis for bimolecular reactions, it cannot explain the trend in rate accelerations shown in Fig. 1. However, a change in the nature of the solvent can increase reaction rates dramatically. Crosby et al. (12Crosby J. Stone R. Lienhard G.E. J. Am. Chem. Soc. 1970; 92: 2891-2900Crossref PubMed Scopus (177) Google Scholar) showed that simply changing the solvent from water to ethanol can increase the rate for a model decarboxylase reaction by 104. Moreover, both experimental and theoretical studies have shown that reactions in solution are often retarded by the solvent when compared with a similar gas phase reaction (13Tanaka K. Mackay G.I. Payzant J.D. Bohme D.K. Can. J. Chem. 1976; 54: 1643-1659Crossref Google Scholar, 14Jorgensen W.L. Adv. Chem. Phys. 1988; 70: 469-488Google Scholar, 15Albery W.J. Annu. Rev. Phys. Chem. 1980; 31: 227-263Crossref Google Scholar, 16Chabynic M.L. Craig S.L. Regan C.K. Brauman J.I. Science. 1998; 279: 1882-1885Crossref PubMed Scopus (264) Google Scholar). Table Icompares rates for theS n2 displacement reaction, CH3Br + Cl− → CH3Cl + Br−, in solvents of differing polarity and dielectric response, including a “null” solvent, the gas phase environment. The effect of the solvent is to retard the rate relative to what would be observed for this reaction under the same conditions in the gas phase. The solvent must reorganize to obtain a configuration that will allow the reaction to proceed (1Cannon W.R. Singleton S.F. Benkovic S.J. Nat. Struct. Biol. 1996; 3: 821-833Crossref PubMed Scopus (134) Google Scholar, 15Albery W.J. Annu. Rev. Phys. Chem. 1980; 31: 227-263Crossref Google Scholar, 17Krishtalik L.I. Mol. Biol. (Mosc.). 1974; 8: 75-81PubMed Google Scholar, 18Warshel A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5250-5254Crossref PubMed Scopus (437) Google Scholar, 19Krishtalik L.I. J. Theor. Biol. 1980; 86: 757-771Crossref PubMed Scopus (42) Google Scholar, 20Northrup S.H. McCammon J.A. J. Am. Chem. Soc. 1984; 106: 930-934Crossref Scopus (32) Google Scholar, 21Krishtalik L.I. Topolev V.V. J. Theor. Biol. 1985; 112: 251-264Crossref PubMed Scopus (23) Google Scholar, 22Yadav A. Jackson R.M. Holbrook J.J. Warshel A. J. Am. Chem. Soc. 1991; 113: 4800-4805Crossref Scopus (110) Google Scholar). The fast rate of reactions in the gas phase led Dewar and Storch (23Dewar M.J.S. Storch D.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2225-2229Crossref PubMed Scopus (199) Google Scholar) to propose that enzymes act through a desolvation mechanism, but it was pointed out by Warshel et al. (24Warshel A. Aqvist J. Creighton S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5820-5824Crossref PubMed Scopus (136) Google Scholar) that the effect is more correctly described as solvent substitution. Early on, Perutz (25Perutz M. Proc. R. Soc. Lond. B Biol. Sci. 1967; 167: 448Crossref Google Scholar) suggested such a scenario, and Krishtalik (17Krishtalik L.I. Mol. Biol. (Mosc.). 1974; 8: 75-81PubMed Google Scholar) and Warshel (18Warshel A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5250-5254Crossref PubMed Scopus (437) Google Scholar) offered detailed explanations. Krishtalik (17Krishtalik L.I. Mol. Biol. (Mosc.). 1974; 8: 75-81PubMed Google Scholar) examined enzyme reactions with a simple yet insightful model that considered the enzyme to be a low dielectric, low polarity medium, whereas Warshel (18Warshel A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5250-5254Crossref PubMed Scopus (437) Google Scholar) made a further step in the model development by including a preorganized polar framework (see the third minireview in this series by Warshel (49Warshel A. J. Biol. Chem. 1998; 273: 27035-27038Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar) for more details).Table IReaction rates for an Sn2 reaction in various solvents of differing dielectric response and polarity, including the gas phase reaction (13Tanaka K. Mackay G.I. Payzant J.D. Bohme D.K. Can. J. Chem. 1976; 54: 1643-1659Crossref Google Scholar)Cl− + CH3Br → ClCH3 + Br−SolventRelative ratecm molecule sGas phase1MeCO10−10DMF10−11CH3OH10−15H2O10−16Dimethylformamide. Open table in a new tab Dimethylformamide. Serum albumin is well known to act as a weak catalyst by a medium effect. Hollfelder et al. (26Hollfelder F. Kirby A.J. Tawfik D.S. Nature. 1997; 383: 60-63Crossref Scopus (157) Google Scholar) have shown that this transport protein can compete with the catalytic antibody 34E4 with respect to rate enhancements (k cat /k non) for an elimination reaction initiated by proton removal. This result is reminiscent of work with subtilisin in which the catalytic triad was replaced by three alanine residues, yet the modified enzyme was still capable of a rate enhancement of 103–104 for anilide hydrolysis even without forming the acyl enzyme intermediate (27Carter P. Wells J.A. Proteins. 1990; 7: 335-342Crossref PubMed Scopus (77) Google Scholar). The effect of the medium on the reaction will depend on both the dielectric response and the polarity of the medium. For example, enzyme active sites generally have a low dielectric response but are highly polar and thus can generate intense electric fields. An active site will retard a reaction rate unless it is organized to be electrostatically complementary to the transition state configuration of the reactant (21Krishtalik L.I. Topolev V.V. J. Theor. Biol. 1985; 112: 251-264Crossref PubMed Scopus (23) Google Scholar, 22Yadav A. Jackson R.M. Holbrook J.J. Warshel A. J. Am. Chem. Soc. 1991; 113: 4800-4805Crossref Scopus (110) Google Scholar). If this is not the case, the reactant can become “locked in” to a nonreactive configuration, that is, enzymes provide more than a simple medium effect. 2As in all generalizations, the concept that biochemically relevant reactions will be slower in aqueous solvent than an organic solvent such as dimethyl formamide (e.g. Table I) has exceptions. For example, the reaction catalyzed by chorismate mutase, the Claisen rearrangement of chorismate to prephenate, is actually 100-fold faster in water than in methanol. However, as one might anticipate, the rate acceleration that the enzyme provides (106) is modest and is on the lower end of the scale of Fig. 1. In this case, the enzyme increases the reaction rate to a large extent by organizing the reactant relative to the configurations found in aqueous or nonaqueous solution. In such cases as this, a catalytic antibody may provide for reaction rates approaching those seen in their enzymatic counterparts (28Haynes M.R. Stura E.A. Hilvert D. Wilson I.A. Science. 1994; 263: 646-652Crossref PubMed Scopus (154) Google Scholar). However, because catalytic antibodies do not contain deep active site clefts and hence will not shield the reaction as well from the effects of bulk solvent, it would be surprising to find catalytic antibodies that approach the rate enhancements seen for the reactions in Fig. 1 with the largest rate enhancements. In contrast to reactions in solution, the enzymic environment is preorganized to be complementary to the transition state configuration of the reactants, and as a result the reorganization penalty is relatively small. As an example consider the possible events that can occur while a reactant is bound to the active site of an enzyme. Protein conformational changes can occur on a wide range of time scales as shown in Table II (29McCammon J.A. Harvey S.C. Dynamics of Proteins and Google Scholar). the fastest are the bond vibrations on the order of to of The which is used in transition state theory occurs on the time time scales of various events that can occur in an enzyme complex (29McCammon J.A. Harvey S.C. Dynamics of Proteins and Google to of to to of to of to energy of D. J. 1991; PubMed Scopus (42) Google to of medium in to to to catalysis Benkovic S.J. PubMed Scopus Google Open table in a new tab In contrast, the of a in the of a protein occurs on the second to time scale S.H. M.R. McCammon J.A. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). this time associated with can occur up to although not all such would result in a reaction. If enzyme catalysis were on a conformational change such as then the step would be the of the effects resulting from bond vibrations would be by the conformational change (1Cannon W.R. Singleton S.F. Benkovic S.J. Nat. Struct. Biol. 1996; 3: 821-833Crossref PubMed Scopus (134) Google Scholar). For the chemical reaction occurs on the time which is time for many conformational changes to occur that could the reaction. If this were the case, then the conformational change effects from faster degrees of freedom such as bond However, the reaction rate under conditions Table a effect of which stems from changes in the nature of a bond We that significant reorganization of the complex is for catalysis to reorganization of the reaction environment can the of a reaction S.H. McCammon J.A. J. Am. Chem. Soc. 1984; 106: 930-934Crossref Scopus (32) Google Scholar). In this a significant of catalysis can be for by a preorganized and reaction environment when compared with reactions in solution. For example, the reactions catalyzed by and decarboxylase have the largest rate accelerations cat /Km)/k non) to (5Radzicka A. Wolfenden R. Science. 1995; 267: 90-93Crossref PubMed Scopus (741) Google Scholar) cat /Km)/k non) = and in Fig. 1). In both cases studies have shown that the model solution reaction rate can be or more of by simply the active site by weak and the dielectric solvent environment with a low dielectric one that is complementary to the proposed transition state of the reactants J. Warshel A. 1989; PubMed Scopus Google Scholar, Science. 1997; PubMed Scopus Google Scholar). in the of it has been determined that the is a and not an essential as Wolfenden R. J. Am. Chem. Soc. 1998; Scopus Google The rate increase for the model reaction is obtained without unusually strong or interactions between the transition state of the reactants and the enzyme. For the uncatalyzed reactions of a simple change in the nature of the environment occurs during the binding can account for rate enhancements. A chemical reaction is an and as such it might be to propose that the reaction within the enzyme active site are from those in solution. effects are for in transition state theory by the use of a which will be much less than if such effects are However, for both relevant solution reactions and enzyme reactions studied to significant from transition state theory = have been seen A. Proc. Natl. Acad. Sci. U. S. A. 1984; PubMed Scopus (157) Google Scholar, A. F. J. Mol. Biol. 1988; PubMed Scopus Google Scholar, M. Chem. Phys. 1997; 267: Scopus Google Scholar). along the reaction does occur for for example, as the between the and increase with the low of the barrier for the reaction also decreases and However, such of the barrier are taken into account in transition state theory by the use of a free energy barrier in which the barrier is a An perspective on of transition state theory to can be found in Science. 1985; PubMed Scopus Google this it is not clear how in enzymes along the reaction a of enzymic are compared, the observed rate enhancements and the dissociation TS can many of However, this does not that those with the of K TS have stronger interactions between the enzyme and the transition state configuration of the the large rate enhancements result from large solvent reorganization required for the reference solution reaction to This is in K TS by an free energy of the reactant in its transition state This is not to say that various aspects of enzyme such as and effects T.C. J. Am. Chem. Soc. 1996; 118: Scopus Google Scholar, T.C. Benkovic S.J. A. Scholar, Science. 1997; PubMed Scopus Google Scholar), hydrogen bond J.A. Kreevoy M.M. Cleland W.W. Frey P.A. Chem. Biol. 1997; Full Text PDF PubMed Scopus Google Scholar, D. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), S.J. J. Am. Chem. Soc. 1996; 118: Scopus Google Scholar), and are not does it that interactions between the substrate and the enzyme are not stronger in in However, as was pointed out years (18Warshel A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5250-5254Crossref PubMed Scopus (437) Google Scholar), we cannot enzymic rate enhancements we solution reactions.
Source: Journal of Biological Chemistry
Authors: William R. Cannon, Stephen J. Benkovic
Institution: Pennsylvania State University
Language: English
Subfield: Organic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1993916927