- Email: [email protected]
29 The Comparison of the Steps of Some Enzyme-Catalyzed and Base-Catalyzed Hydrolysis Reactions
29
The Comparison of the Steps of Some Enzyme-Catalyzed and Base-Catalyzed Hydrolysis Reactions H. GUTFREUND Department of Colloid Science, University of Cambridge, England
It has been shown that enzyme-catalyzed hydrolysis reactions, especially those involving trypsin, chymotrypsin, and some plant peptidases, proceed through a number of well-defined steps. The first of these steps is a rapid second-order reaction, which is thought to be an adsorption of the substrate on to the “specificity site” of the enzyme. Subsequent first-order reactions involve two or more basic groups of the “catalytic site” of the enzyme molecule and the carbonyl carbon of the substrate. Two pre-steady-state methods for the study of consecutive steps in enzyme-catalyzed reactions are described. The first involves the initial acceleration of the rate of formation of the final products, and the second the observation of reaction intermediates. Some results of the application of these methods to the characterization of intermediate steps in several hydrolysis reactions, as well as a model for the path of such enzyme reactions, are given. This model is based on the identification of the basic groups on the catalytic sites and can be extended to explain the nature of transfer reactions. The kinetic consequences of such a sequence of reaction steps and their contribution to the efficiency of enzyme reactions as compared with homogeneous base-catalyzed reactions are discussed.
I. INTRODUCTION Enzymes play the dual role of selecting-by means of their specificity -one of a number of reaction paths and accelerating the chosen one. The theory for the mechanisms of some enzyme-catalyzed reactions which is developed here is based on a definite kinetic scheme which is an extension of the well-known Michaelis-Menten hypothesis :
+
E +SeES--+E P (1) where E , S, ES, and P represent enzyme, substrate, compound, and product, respectively. Such a complex reaction will involve more than one form of intermediate enzyme-substrate compound. Some of the kinetic and equilibrium consequences of certain aspects of the multiplicity of enzyme-substrate compounds have been analyzed by Foster and Niemann 284
29.
ENZYME-CATALYZED HYDROLYSIS REACTIONS
285
(I), and some evidence for two kinetically distinct intermediates has been obtained by Wilson and Calib (2) and by Smith, Finkle, and Stockell (3). It is the general purpose of the investigations reported here to develop methods which give definite and quantitative evidence for some steps in the reaction of substrate with enzyme and for the existence of distinct enzyme-substrate compounds and to use such information to identify the chemical nature of such steps. Experimental work by the present author has been restricted to the reactions of some pure protein enzymes, which do not require prosthetic groups or coenzymes for their activity. It will be seen, however, that the methods used may be very powerful for the analysis of the sequence of reaction steps in more complex enzyme-coenzyme-substrate systems. 11. KINETICPROCEDURES 1. Steady-State Data
The two important kinetic results obtained from studies of the steady state of enzyme-catalyzed reactions are the Michaelis constant K M and the maximum velocity Vmax.These constants are determined from one of a number of graphical procedures relating the initial velocity VOto the initial substrate concentration [SlO over a range of [Sl0.They are related by the well-known expression
and characterize an enzyme system under a particular set of physical conditions, Foster and Niemann ( 1 ) have pointed out that the effects of changes of conditions (substrate structure, pH, temperature, etc.) on the over-all steady-state velocity have to be interpreted with care and that they do not necessarily give information about changes in one specific rate constant but may be dependent upon the equilibria involved in the formation of the several enzyme-substrate intermediates. In the Michaelis-Menten scheme, K , gives the steady-state concentration of ES,
and V,, = k[E]o,where [Elo is the total enzyme concentration and k the rate of decomposition of the enzyme substrate complex. An analysis of the kinetics of an enzyme reaction proceeding through a number of steps shows that under different conditions K , and k can apply to different rate-determining steps or to a combination of them. One can develop kinetic equations of a general type which would describe the behavior of
286
H . GUTFREUND
an enzyme-catalyzed reaction consisting of one second-order step and n consecutive first-order reaction steps. This does not, however, appear to be very useful for the purpose of translating experimental results into a physical model of the reaction mechanism. So far, experimental evidence from “pure protein” enzyme systems has given concrete evidence for only two intermediate compounds; the Michaelis-Menten scheme is therefore used in the following extended form:
Kinetic equations derived for such a scheme have the merit of having been found of practical application to the interpretation of experimental results, which compensates for their lack of generality. 2. Applications of Pre-Steady-State Kinetics
If enzymes and substrate undergo a series of reactions, the first of these will be a second-order reaction, while all subsequent steps will be first order. This argument is used throughout either to prove that a particular step studied must be the true initial enzyme-substrate combination or in other cases to demonstrate that some particular intermediate step, which follows first-order kinetics, must have been preceded by a second-order initial compound formation. Enzyme reactions involving prosthetic groups or coenzymes can often be studied by observation of the spectral changes which occur during compound formation. So far, no such spectral changes have been observed in pure protein enzyme systems, and for this purpose two other methods have been developed for the study of steps in the formation and decomposition of enzyme-substrate compounds (4). The first of these, the “initial acceleration method” can be used for the study of the second-order reaction, which is visualized as a rapid adsorption of the substrate on the “specificity site” of the enzyme. The second procedure relies on observable intermediates such as are obtained when spectral changes in the substrate occur or when several products are liberated from the enzyme-substrate compound at different stages of the reaction. Both methods have been used with the Gibson (5) stopped-flow apparatus, which was slightly modified (4, 6) for some special applications. The use of the stopped-flow methods in the study of enzyme reaction mechanisms was derived from Chance’s classical work on catalase and peroxidase and on the sequence of events in biological oxidation reactions (7). The applications of these techniques which are described here are, however, inherently different from Chance’s approach.
29.
ENZYME-CATALYZED
HYDROLYSIS REACTIONS
287
3. Initial Acceleration
The initial accelerntion of enzyme reactions can be observed by a study of the rate of appearance of the final product during the short time interval between mixing of enzyme and substrate and the attainment of the steadystate concentrations of all the intermediate compounds. Apart from the final steady-state velocity, this method can, in principle, give information about the kinetics of two reaction steps. In the first place, the second-order constant k l which characterizes the initial enzyme-substrate combination can be determined when [Sl0, the initial substrate concentration, is sufficiently small to make this step rate-determining during the pre-steadystate period. Kinetic equations for the evaluation of rate constants from pre-steady-state data have recently been derived (4). Under suitable conditions kl can be evaluated from where r is the intercept on the time axis obtained when the steady-state rate is extrapolated to [PI = 0. Secondly, a t high substrate concentrations, when k2 can become rate-determining for the initial acceleration, This method of studying the pre-steady-state kinetics of enzyme-catalyzed reactions has given some interesting results (4, 8). In many cases, the initial enzyme-substrate combination is very rapid. With the techniques available at present, only the lower limit k l > 2 X lo6 em.-’ see.-’ could be determined for the reactions of chymotrypsin and trypsin with their respective amino acid ester substrates. The rate of the initial enzymesubstrate combination for the reaction of the plant peptidase ficin with benzoyl-L-arginine ethyl ester was found to be comparatively slow, k1 = 5 X lo2 cm.-’ set.?. It was shown (4) that this reaction followed secondorder kinetics.
4. Observable Intermediates Photometric observation of the change in concentration of spectroscopically distinct substrates, intermediates, or products during the pre-steadystate phase of enzyme reactions is the most promising procedure for obtaining detailed information about the sequence of steps in such reactions. So far we have applied this method only to enzyme-catalyzed hydrolysis reactions. This can be done in two ways: in the first place, if the reaction mixture contains an indicator color, the liberation or binding of hydrogen ions during the course of the reaction can be followed. Secondly, we have studied the hydrolysis of a number of nitrophenyl esters, and we have
288
H. GUTFREUND
found that the liberation of the colored nitrophenylate ion and of the acylating group from the enzyme-substrate compound can be followed independently (6). For the interpretation of these observations, Gutfreund and Sturtevant (6) have derived expressions for the steady-state rate and Michaelis' constants in terms of the individual rate constants of the threestage process described in equation (2). The expressions are
1
1
Ic-k,
+ k3-1
(4)
Equations (3) and (4) are based on the assumption that the reversal of the second and third steps can be neglected; this is always true when initial rate measurements are used. Applications of the three-step kinetic equations to hydrolysis and acyl transfer reactions will be seen in the following sections. Further applications of this approach to many enzyme reactions are planned with the use of ultraviolet spectroscopy for the detection of intermediates during the pre-steady-state phase. 111. MODELSOF ENZYME MECHANISMS 1. The Mechanism of the Reactions of Chymotrypsin and Similar Enzymes
We have suggested (9, 10) that both in trypsin- and chymotrypsincatalyzed ester hydrolysis the rate-determining step is dependent on an imidazole group in its basic form on the enzyme molecule. The model first proposed involved only two kinetically distinguishable steps : they are the rapid initial adsorption of the substrate on the specificity site of the enzyme and the subsequent rate-determining attack of the imidazole group of the catalytic site on the carbonyl carbon of the substrate. This model had some shortcomings, and it was possible to eliminate these when the results of more recent experiments suggested an extension of the above scheme. It was found by Hartley and Kilby (11) that chymotrypsin catalyzes the hydrolysis of p-nitrophenyl acetate. From a study of the kinetics of the hydrolysis of p-nitrophenyl acetate (6) and 2,4-dinitro-phenyl acetate (8) by the stopped-flow technique, we could distinguish three steps in these reactions: first, an initial fast adsorption of the substrate, second, a liberation of one mole of nitrophenol per mole of chymotrypsin and a concomitant acylation of a group on the enzyme, and, third, the hydrolysis of the enzyme-acyl compound. The initial adsorption step is too fast to be measured by the method available at present. Because of their relative magnitudes, the subsequent two steps characterized by kz = 3 set.-' and k, = 0.025
29.
ENZYME-CATALYZED HYDROLYSIS REACTIONS
289
set.-', which are involved in the chemical reaction between groups on the catalytic site and the substrate, could be analyzed separately and in detail. Both the liberation of nitrophenylate ions and the liberation or binding of hydrogen ions were followed during the course of the two consecutive reactions. It was found that during the first of these only nitrophenol was liberated, while the acetate reacts with an OH group of the enzyme. This acyl-enzyme is hydrolyzed, with liberation of acetate, during the final step. Both'the acylation and the hydrolysis of the acyl-enzyme are inhibited by the protonation of a basic group, probably an imidazole group, in the vicinity. We made the interesting observation (8) that this basic group changes its pK during the acylation reaction, thus giving the two steps a different pH dependence. We have surveyed the considerable volume of evidence (6, 8) that it is the OH group of a serine residue of chymotrypsin which becomes acylated. There is much evidence that everything that has been said above about chymotrypsin is also true for trypsin. We have shown by a comparison of the pH dependence of the step characterized by kz that the hydrolysis of the enzyme-acyl compound is the rate-determining step for the enzymatic hydrolysis of the usual amino acid amide substrates. In the case of chymotrypsin, acetyl-L-phenylalanine ethyl ester is hydrolyzed 1,000 times faster than the corresponding amide; and in the case of trypsin, benzoyl-L-arginine ethyl ester is hydrolyzed 300 times faster than the corresponding amide. This suggests that for the amide hydrolysis too the second step, the acylation of the enzyme, must be the rate-determining step, since the third step is obviously identical for esters and amides of the same amino acid derivatives. The pH dependence of the chymotrypsin-catalyzed hydrolysis of acetyl-L-tyrosine ethyl ester and acetyl-L-phenylalanine ethyl ester indicates that for these reactions kz and k3 are of the same order of magnitude and both contribute to the over-all rate, as shown by Equation (4). 2. The Mechanism of the Reactions of Ficin and Similar Enzymes There are two distinct classes of hydrolytic enzymes: those which have a reduced S H group as part of their active center and others which do not have such a group. Trypsin and chymotrypsin are among the latter, while ficin and papain are among the former. We have taken up the study of ficin-catalyzed reactions side by side with our studies on trypsin because it was obvious that the two enzymes catalyze the same reaction via a different mechanism. On comparing our results for ficin (4, 12) with those of Smith, Finkle, and Stockell (3) as well as with some of our own on papain, we find that from the point of view of kinetics and mechanism they appear to be very closely related enzymes. In the subsequent discussion, we assume that all that is said about ficin applies equally to papain and probably also to other plant --SH peptidases.
290
H . GUTFREUND
From our studies of the inhibition of ficin by methyl-mercury we know that one -SH group is required for the activity of the enzyme. It has not yet been possible to obtain spectroscopic or other conclusive evidence that a thiol ester between this S H group and the acidic part of the substrate is formed during one step of the catalytic hydrolysis, but there is much indirect evidence that this is the case. We postulate a three-stage mechanism for ficin-catalyzed reactions similar to that suggested for trypsin and chymotrypsin. First, a rapid adsorption step, second, the acylation of the S H group (in place of the OH group suggested for the other enzymes), and, third, the hydrolysis of the enzyme-acyl compound (thiol ester). Studies of the effect of pH, temperature, and solvent composition on this third step indicate that an ionized carboxyl group controls its rate. Kinetic data show that in the case of ficin the enzyme-acyl compound is more stable than it is in the case of trypsin or chymotrypsin. This has two interesting consequences: first, it makes ficin a more efficient enzyme for transfer reactions-this will be discussed in the next section-and, secondly, it hydrolyzes esters and amides a t nearly the same rate. I n our three-stage scheme, k, = 1.5 set.-' is the same for ester and amide substrates and is rate-determining for the ester hydrolysis. The over-all rate for the ester hydrolysis is determined by k~ , while the over-all rate of the amide hydrolysis is characterized by a rate constant k = 0.65 sec.,-l which must be a function of kz and k, [see Equation (4)]. Suitable substrates for a separate investigation of the second and third step of ficin-catalyzed reaction are being examined at present. 3. The Mechanism of Transfer Reactions
It has been demonstrated that most hydrolytic enzymes catalyze a large variety of reactions of the carbonyl group of their specific substrate. The most interesting of these reactions involve acyl transfer. A typical example is the reaction studied by Durell and Fruton (13):
Benzoyl-arginine amide
+
H20
7
Benzoyl-arginine
NHzOH
L
+ NHl+
Benzoyl-arginine hydroxamic acid
+ NH4+
Enzymatic transfer of phosphate is also of great interest, and examples and a proposed mechanism have been given by Morton (14). All reactions of hydrolytic enzyme will involve the acyl-enzyme formation proposed above, and the subsequent step will depend on whether the acyl-enzyme reacts with water to give the hydrolysis products or with another nucleophilic reagent to form the acyl-transfer product.
29.
ENZYME-CATALYZED HYDROLYSIS
REACTIONS
291
For the example of hydroxamic acid formation given above, the efficiency of the exchange reaction depends on the relative nucleophilic strength of HzO and NHzOH and on the concentration of the latter. Hydroxylamine is a stronger nucleophilic replacement reagent but is present in relatively low concentration; it is therefore favored by the more stable enayme-acyl substrate bond. We have shown that the stability of the acylated enzyme is characterized by JC3 and that for comparable reactions of trypsin and ficin on papain the rate of hydrolysis of the acyl-enzyme compound of the former is ten times as fast as that of the latter. The findings of Durell and Fruton ( I S ) that papain is ten times asefficient as a transfer enzyme than trypsin is in good agreement with the proposed scheme. From a biochemical point of view, it is of great interest to know which of the enzymes studied mainly for their hydrolytic activity are capable of catalyzing the synthesis of various amide, peptide, ester, and similar bonds and whether they actually do so in biological systems. Far too little is known about this at present to make any significant generalizations, but one can see that an enzyme which will catalyze hydrolysis reactions under one condition will catalyze synthesis under other conditions. For instance, a small change of pH can decrease the rate of decomposition of the enzymeacyl compound without changing the rate of its formation from enzyme and substrate and can thus favor nonhydrolytic transfer reactions. Studies of the effect of pH on transfer have often been obscured by the concomitant change in the ionization of the acceptors, and this very interesting field of enzyme catalysis requires a great deal of further detailed investigation. One other interesting point arises from a consideration of the thermodynamic aspects of transfer reactions. Biochemists call an ester or anhydride bond with a large positive free energy of formation an “energy-rich” bond, and such energy-rich compounds take an active and varied part in biological transfer reactions. The enzyme-acyl substrate bonds may well be regarded as high up in the scale of “energy-rich” bonds. The free energy of adsorption in the initial enzyme-substrate compound formation would contribute to the formation of a compound with a high free-energy content.
IV. THE EFFICIENCY OF ENZYME-CATALYZED REACTIONS Enzyme-catalyzed hydrolysis reactions of derivatives of relatively complex compounds such as amino acids, sugars, nucleotides, etc., involve one step which is absent in all base-catalyzed reactions, that is, the initial adsorption of the specific residue on the specificity site of the enzyme. We have shown that this rapid adsorption step precedes the chemical interaction between the catalytic site of the enzyme and the susceptible group of the substrate, and it is possible to see in a qualitative manner that the first step will aid the second one. For certain models one can make a quantitative assessment of the“spec-
292
H. GUTFREUND
ificity binding” contribution to the efficiency of enzyme catalysis. If one basic group, say, an imidazole group, were the sole constituent of the catalytic site, one could compare the known catalytic activity of imidazole derivatives in homogeneous hydrolysis reactions with that of the enzyme, It is probably justifiable to compare the first-order constant ks of the enzyme reaction
with k , the first-order constant of the homogeneous catalyzed reaction. The rates of the reactions characterized by kS and k , are dependent on enzyme concentration and on catalyst concentration, respectively. The apparent free energy of activation AFZf calculated from the first-order kinetics of enzyme-catalyzed reactions is given by AF,’
= AFt
- AFB
where AFt is the free energy of activation of the reaction characterized by k and AFB is the free energy of binding substrate to enzyme as determined by K , . If AFT is similar to the free energy of activation of the homogeneous reaction catalyzed by such a group, then the numerical contribution of the free energy of binding is clear. However, the model of enzyme-catalyzed hydrolysis reactions presented here has an additional degree of complication, since the binding of the substrate not only brings the catalytic group of the enzyme (imidazole) into the vicinity of the reactive part of the substrate, but also brings another group of the enzyme into such a position that it forms an acyl compound with the acidic part of the substrate. Studies with model compounds simulating such a situation have not yet gone very far. It may perhaps be wise to await the maximum amount of detail which can be obtained from studies of the enzyme mechanisms before embarking on the difficult task of making suitable models. It is not very surprising that the evaluation of the heats of activation of various enzyme-catalyzed ester hydrolysis reactions has proved to be uninformative, the values being very close to those of base-catalyzed hydrolysis of esters. It may, however, prove very useful when the work on the differentiation between separate steps, described in this paper, can be extended by a study of the effect of temperature on the different steps.
ACKNOWLEDGMENT Much of the work described in this paper was carried out in collaboration with Dr. Julian M. Sturtevant, whose constant advice and help in many ways is gratefully acknowledged.
Received: April 6 , 1966 (in revised f o m June 18, 1956).
29.
ENZYME-CATALYZED HYDROLYSIS REACTIONS
293
REFERENCES I . Foster, R. J., and Niemann, C., Proc. Natl. Acad. Sci. (U.S.) 39, 371 (1956). 2. Wilson, I. B., and Calib, E., J. Am. Chem. SOC.78, 202 (1956). 3. Smith, Emil L., Finkle, B. J., and Stockell, A., Discussions Faraday Soc. NO. 20, 96 (1955). 4. Gutfreund, H., Discussions Faraday SOC.No. 20, 167 (1955). 6. Gibson, Q. H., Discussions Faraday,Soc. No. 17, 137 (1954). 6. Gutfreund, H., and Sturtevant, J. M., Biochem. J . 63, 656 (1956). 7. Chance, B., in “The Mechanism of Enzyme Action” (W. D. McElroy and B. Glass, eds.), p. 399. John Hopkins Press, Baltimore, 1954. 8. Gutfreund, H., and Sturtevant, J. M., Proc. Natl. Acad. Sci. (U.S.)42, 719 (1956). 9. Gutfreund, H., Trans. Faradag SOC.61, 441 (1955). 10. Hammond, B. R., and Gutfreund, H., Biochem. J. 61, 187 (1955). 11. Hartley, B. S., and Kilby, B. A., Biochem. J . 60,672 (1952). fb. Bernhard, S. A., and Gutfreund, H., Biochem. J. 63, 61 (1956). 18. Durell, J., and Fruton, J. S., J. Biol. Chem. 207, 497 (1954). 1.4. Morton, R. K., Nature 172, 65 (1953).
The Comparison of the Steps of Some Enzyme-Catalyzed and Base-Catalyzed Hydrolysis Reactions H. GUTFREUND Department of Colloid Science, University of Cambridge, England
It has been shown that enzyme-catalyzed hydrolysis reactions, especially those involving trypsin, chymotrypsin, and some plant peptidases, proceed through a number of well-defined steps. The first of these steps is a rapid second-order reaction, which is thought to be an adsorption of the substrate on to the “specificity site” of the enzyme. Subsequent first-order reactions involve two or more basic groups of the “catalytic site” of the enzyme molecule and the carbonyl carbon of the substrate. Two pre-steady-state methods for the study of consecutive steps in enzyme-catalyzed reactions are described. The first involves the initial acceleration of the rate of formation of the final products, and the second the observation of reaction intermediates. Some results of the application of these methods to the characterization of intermediate steps in several hydrolysis reactions, as well as a model for the path of such enzyme reactions, are given. This model is based on the identification of the basic groups on the catalytic sites and can be extended to explain the nature of transfer reactions. The kinetic consequences of such a sequence of reaction steps and their contribution to the efficiency of enzyme reactions as compared with homogeneous base-catalyzed reactions are discussed.
I. INTRODUCTION Enzymes play the dual role of selecting-by means of their specificity -one of a number of reaction paths and accelerating the chosen one. The theory for the mechanisms of some enzyme-catalyzed reactions which is developed here is based on a definite kinetic scheme which is an extension of the well-known Michaelis-Menten hypothesis :
+
E +SeES--+E P (1) where E , S, ES, and P represent enzyme, substrate, compound, and product, respectively. Such a complex reaction will involve more than one form of intermediate enzyme-substrate compound. Some of the kinetic and equilibrium consequences of certain aspects of the multiplicity of enzyme-substrate compounds have been analyzed by Foster and Niemann 284
29.
ENZYME-CATALYZED HYDROLYSIS REACTIONS
285
(I), and some evidence for two kinetically distinct intermediates has been obtained by Wilson and Calib (2) and by Smith, Finkle, and Stockell (3). It is the general purpose of the investigations reported here to develop methods which give definite and quantitative evidence for some steps in the reaction of substrate with enzyme and for the existence of distinct enzyme-substrate compounds and to use such information to identify the chemical nature of such steps. Experimental work by the present author has been restricted to the reactions of some pure protein enzymes, which do not require prosthetic groups or coenzymes for their activity. It will be seen, however, that the methods used may be very powerful for the analysis of the sequence of reaction steps in more complex enzyme-coenzyme-substrate systems. 11. KINETICPROCEDURES 1. Steady-State Data
The two important kinetic results obtained from studies of the steady state of enzyme-catalyzed reactions are the Michaelis constant K M and the maximum velocity Vmax.These constants are determined from one of a number of graphical procedures relating the initial velocity VOto the initial substrate concentration [SlO over a range of [Sl0.They are related by the well-known expression
and characterize an enzyme system under a particular set of physical conditions, Foster and Niemann ( 1 ) have pointed out that the effects of changes of conditions (substrate structure, pH, temperature, etc.) on the over-all steady-state velocity have to be interpreted with care and that they do not necessarily give information about changes in one specific rate constant but may be dependent upon the equilibria involved in the formation of the several enzyme-substrate intermediates. In the Michaelis-Menten scheme, K , gives the steady-state concentration of ES,
and V,, = k[E]o,where [Elo is the total enzyme concentration and k the rate of decomposition of the enzyme substrate complex. An analysis of the kinetics of an enzyme reaction proceeding through a number of steps shows that under different conditions K , and k can apply to different rate-determining steps or to a combination of them. One can develop kinetic equations of a general type which would describe the behavior of
286
H . GUTFREUND
an enzyme-catalyzed reaction consisting of one second-order step and n consecutive first-order reaction steps. This does not, however, appear to be very useful for the purpose of translating experimental results into a physical model of the reaction mechanism. So far, experimental evidence from “pure protein” enzyme systems has given concrete evidence for only two intermediate compounds; the Michaelis-Menten scheme is therefore used in the following extended form:
Kinetic equations derived for such a scheme have the merit of having been found of practical application to the interpretation of experimental results, which compensates for their lack of generality. 2. Applications of Pre-Steady-State Kinetics
If enzymes and substrate undergo a series of reactions, the first of these will be a second-order reaction, while all subsequent steps will be first order. This argument is used throughout either to prove that a particular step studied must be the true initial enzyme-substrate combination or in other cases to demonstrate that some particular intermediate step, which follows first-order kinetics, must have been preceded by a second-order initial compound formation. Enzyme reactions involving prosthetic groups or coenzymes can often be studied by observation of the spectral changes which occur during compound formation. So far, no such spectral changes have been observed in pure protein enzyme systems, and for this purpose two other methods have been developed for the study of steps in the formation and decomposition of enzyme-substrate compounds (4). The first of these, the “initial acceleration method” can be used for the study of the second-order reaction, which is visualized as a rapid adsorption of the substrate on the “specificity site” of the enzyme. The second procedure relies on observable intermediates such as are obtained when spectral changes in the substrate occur or when several products are liberated from the enzyme-substrate compound at different stages of the reaction. Both methods have been used with the Gibson (5) stopped-flow apparatus, which was slightly modified (4, 6) for some special applications. The use of the stopped-flow methods in the study of enzyme reaction mechanisms was derived from Chance’s classical work on catalase and peroxidase and on the sequence of events in biological oxidation reactions (7). The applications of these techniques which are described here are, however, inherently different from Chance’s approach.
29.
ENZYME-CATALYZED
HYDROLYSIS REACTIONS
287
3. Initial Acceleration
The initial accelerntion of enzyme reactions can be observed by a study of the rate of appearance of the final product during the short time interval between mixing of enzyme and substrate and the attainment of the steadystate concentrations of all the intermediate compounds. Apart from the final steady-state velocity, this method can, in principle, give information about the kinetics of two reaction steps. In the first place, the second-order constant k l which characterizes the initial enzyme-substrate combination can be determined when [Sl0, the initial substrate concentration, is sufficiently small to make this step rate-determining during the pre-steadystate period. Kinetic equations for the evaluation of rate constants from pre-steady-state data have recently been derived (4). Under suitable conditions kl can be evaluated from where r is the intercept on the time axis obtained when the steady-state rate is extrapolated to [PI = 0. Secondly, a t high substrate concentrations, when k2 can become rate-determining for the initial acceleration, This method of studying the pre-steady-state kinetics of enzyme-catalyzed reactions has given some interesting results (4, 8). In many cases, the initial enzyme-substrate combination is very rapid. With the techniques available at present, only the lower limit k l > 2 X lo6 em.-’ see.-’ could be determined for the reactions of chymotrypsin and trypsin with their respective amino acid ester substrates. The rate of the initial enzymesubstrate combination for the reaction of the plant peptidase ficin with benzoyl-L-arginine ethyl ester was found to be comparatively slow, k1 = 5 X lo2 cm.-’ set.?. It was shown (4) that this reaction followed secondorder kinetics.
4. Observable Intermediates Photometric observation of the change in concentration of spectroscopically distinct substrates, intermediates, or products during the pre-steadystate phase of enzyme reactions is the most promising procedure for obtaining detailed information about the sequence of steps in such reactions. So far we have applied this method only to enzyme-catalyzed hydrolysis reactions. This can be done in two ways: in the first place, if the reaction mixture contains an indicator color, the liberation or binding of hydrogen ions during the course of the reaction can be followed. Secondly, we have studied the hydrolysis of a number of nitrophenyl esters, and we have
288
H. GUTFREUND
found that the liberation of the colored nitrophenylate ion and of the acylating group from the enzyme-substrate compound can be followed independently (6). For the interpretation of these observations, Gutfreund and Sturtevant (6) have derived expressions for the steady-state rate and Michaelis' constants in terms of the individual rate constants of the threestage process described in equation (2). The expressions are
1
1
Ic-k,
+ k3-1
(4)
Equations (3) and (4) are based on the assumption that the reversal of the second and third steps can be neglected; this is always true when initial rate measurements are used. Applications of the three-step kinetic equations to hydrolysis and acyl transfer reactions will be seen in the following sections. Further applications of this approach to many enzyme reactions are planned with the use of ultraviolet spectroscopy for the detection of intermediates during the pre-steady-state phase. 111. MODELSOF ENZYME MECHANISMS 1. The Mechanism of the Reactions of Chymotrypsin and Similar Enzymes
We have suggested (9, 10) that both in trypsin- and chymotrypsincatalyzed ester hydrolysis the rate-determining step is dependent on an imidazole group in its basic form on the enzyme molecule. The model first proposed involved only two kinetically distinguishable steps : they are the rapid initial adsorption of the substrate on the specificity site of the enzyme and the subsequent rate-determining attack of the imidazole group of the catalytic site on the carbonyl carbon of the substrate. This model had some shortcomings, and it was possible to eliminate these when the results of more recent experiments suggested an extension of the above scheme. It was found by Hartley and Kilby (11) that chymotrypsin catalyzes the hydrolysis of p-nitrophenyl acetate. From a study of the kinetics of the hydrolysis of p-nitrophenyl acetate (6) and 2,4-dinitro-phenyl acetate (8) by the stopped-flow technique, we could distinguish three steps in these reactions: first, an initial fast adsorption of the substrate, second, a liberation of one mole of nitrophenol per mole of chymotrypsin and a concomitant acylation of a group on the enzyme, and, third, the hydrolysis of the enzyme-acyl compound. The initial adsorption step is too fast to be measured by the method available at present. Because of their relative magnitudes, the subsequent two steps characterized by kz = 3 set.-' and k, = 0.025
29.
ENZYME-CATALYZED HYDROLYSIS REACTIONS
289
set.-', which are involved in the chemical reaction between groups on the catalytic site and the substrate, could be analyzed separately and in detail. Both the liberation of nitrophenylate ions and the liberation or binding of hydrogen ions were followed during the course of the two consecutive reactions. It was found that during the first of these only nitrophenol was liberated, while the acetate reacts with an OH group of the enzyme. This acyl-enzyme is hydrolyzed, with liberation of acetate, during the final step. Both'the acylation and the hydrolysis of the acyl-enzyme are inhibited by the protonation of a basic group, probably an imidazole group, in the vicinity. We made the interesting observation (8) that this basic group changes its pK during the acylation reaction, thus giving the two steps a different pH dependence. We have surveyed the considerable volume of evidence (6, 8) that it is the OH group of a serine residue of chymotrypsin which becomes acylated. There is much evidence that everything that has been said above about chymotrypsin is also true for trypsin. We have shown by a comparison of the pH dependence of the step characterized by kz that the hydrolysis of the enzyme-acyl compound is the rate-determining step for the enzymatic hydrolysis of the usual amino acid amide substrates. In the case of chymotrypsin, acetyl-L-phenylalanine ethyl ester is hydrolyzed 1,000 times faster than the corresponding amide; and in the case of trypsin, benzoyl-L-arginine ethyl ester is hydrolyzed 300 times faster than the corresponding amide. This suggests that for the amide hydrolysis too the second step, the acylation of the enzyme, must be the rate-determining step, since the third step is obviously identical for esters and amides of the same amino acid derivatives. The pH dependence of the chymotrypsin-catalyzed hydrolysis of acetyl-L-tyrosine ethyl ester and acetyl-L-phenylalanine ethyl ester indicates that for these reactions kz and k3 are of the same order of magnitude and both contribute to the over-all rate, as shown by Equation (4). 2. The Mechanism of the Reactions of Ficin and Similar Enzymes There are two distinct classes of hydrolytic enzymes: those which have a reduced S H group as part of their active center and others which do not have such a group. Trypsin and chymotrypsin are among the latter, while ficin and papain are among the former. We have taken up the study of ficin-catalyzed reactions side by side with our studies on trypsin because it was obvious that the two enzymes catalyze the same reaction via a different mechanism. On comparing our results for ficin (4, 12) with those of Smith, Finkle, and Stockell (3) as well as with some of our own on papain, we find that from the point of view of kinetics and mechanism they appear to be very closely related enzymes. In the subsequent discussion, we assume that all that is said about ficin applies equally to papain and probably also to other plant --SH peptidases.
290
H . GUTFREUND
From our studies of the inhibition of ficin by methyl-mercury we know that one -SH group is required for the activity of the enzyme. It has not yet been possible to obtain spectroscopic or other conclusive evidence that a thiol ester between this S H group and the acidic part of the substrate is formed during one step of the catalytic hydrolysis, but there is much indirect evidence that this is the case. We postulate a three-stage mechanism for ficin-catalyzed reactions similar to that suggested for trypsin and chymotrypsin. First, a rapid adsorption step, second, the acylation of the S H group (in place of the OH group suggested for the other enzymes), and, third, the hydrolysis of the enzyme-acyl compound (thiol ester). Studies of the effect of pH, temperature, and solvent composition on this third step indicate that an ionized carboxyl group controls its rate. Kinetic data show that in the case of ficin the enzyme-acyl compound is more stable than it is in the case of trypsin or chymotrypsin. This has two interesting consequences: first, it makes ficin a more efficient enzyme for transfer reactions-this will be discussed in the next section-and, secondly, it hydrolyzes esters and amides a t nearly the same rate. I n our three-stage scheme, k, = 1.5 set.-' is the same for ester and amide substrates and is rate-determining for the ester hydrolysis. The over-all rate for the ester hydrolysis is determined by k~ , while the over-all rate of the amide hydrolysis is characterized by a rate constant k = 0.65 sec.,-l which must be a function of kz and k, [see Equation (4)]. Suitable substrates for a separate investigation of the second and third step of ficin-catalyzed reaction are being examined at present. 3. The Mechanism of Transfer Reactions
It has been demonstrated that most hydrolytic enzymes catalyze a large variety of reactions of the carbonyl group of their specific substrate. The most interesting of these reactions involve acyl transfer. A typical example is the reaction studied by Durell and Fruton (13):
Benzoyl-arginine amide
+
H20
7
Benzoyl-arginine
NHzOH
L
+ NHl+
Benzoyl-arginine hydroxamic acid
+ NH4+
Enzymatic transfer of phosphate is also of great interest, and examples and a proposed mechanism have been given by Morton (14). All reactions of hydrolytic enzyme will involve the acyl-enzyme formation proposed above, and the subsequent step will depend on whether the acyl-enzyme reacts with water to give the hydrolysis products or with another nucleophilic reagent to form the acyl-transfer product.
29.
ENZYME-CATALYZED HYDROLYSIS
REACTIONS
291
For the example of hydroxamic acid formation given above, the efficiency of the exchange reaction depends on the relative nucleophilic strength of HzO and NHzOH and on the concentration of the latter. Hydroxylamine is a stronger nucleophilic replacement reagent but is present in relatively low concentration; it is therefore favored by the more stable enayme-acyl substrate bond. We have shown that the stability of the acylated enzyme is characterized by JC3 and that for comparable reactions of trypsin and ficin on papain the rate of hydrolysis of the acyl-enzyme compound of the former is ten times as fast as that of the latter. The findings of Durell and Fruton ( I S ) that papain is ten times asefficient as a transfer enzyme than trypsin is in good agreement with the proposed scheme. From a biochemical point of view, it is of great interest to know which of the enzymes studied mainly for their hydrolytic activity are capable of catalyzing the synthesis of various amide, peptide, ester, and similar bonds and whether they actually do so in biological systems. Far too little is known about this at present to make any significant generalizations, but one can see that an enzyme which will catalyze hydrolysis reactions under one condition will catalyze synthesis under other conditions. For instance, a small change of pH can decrease the rate of decomposition of the enzymeacyl compound without changing the rate of its formation from enzyme and substrate and can thus favor nonhydrolytic transfer reactions. Studies of the effect of pH on transfer have often been obscured by the concomitant change in the ionization of the acceptors, and this very interesting field of enzyme catalysis requires a great deal of further detailed investigation. One other interesting point arises from a consideration of the thermodynamic aspects of transfer reactions. Biochemists call an ester or anhydride bond with a large positive free energy of formation an “energy-rich” bond, and such energy-rich compounds take an active and varied part in biological transfer reactions. The enzyme-acyl substrate bonds may well be regarded as high up in the scale of “energy-rich” bonds. The free energy of adsorption in the initial enzyme-substrate compound formation would contribute to the formation of a compound with a high free-energy content.
IV. THE EFFICIENCY OF ENZYME-CATALYZED REACTIONS Enzyme-catalyzed hydrolysis reactions of derivatives of relatively complex compounds such as amino acids, sugars, nucleotides, etc., involve one step which is absent in all base-catalyzed reactions, that is, the initial adsorption of the specific residue on the specificity site of the enzyme. We have shown that this rapid adsorption step precedes the chemical interaction between the catalytic site of the enzyme and the susceptible group of the substrate, and it is possible to see in a qualitative manner that the first step will aid the second one. For certain models one can make a quantitative assessment of the“spec-
292
H. GUTFREUND
ificity binding” contribution to the efficiency of enzyme catalysis. If one basic group, say, an imidazole group, were the sole constituent of the catalytic site, one could compare the known catalytic activity of imidazole derivatives in homogeneous hydrolysis reactions with that of the enzyme, It is probably justifiable to compare the first-order constant ks of the enzyme reaction
with k , the first-order constant of the homogeneous catalyzed reaction. The rates of the reactions characterized by kS and k , are dependent on enzyme concentration and on catalyst concentration, respectively. The apparent free energy of activation AFZf calculated from the first-order kinetics of enzyme-catalyzed reactions is given by AF,’
= AFt
- AFB
where AFt is the free energy of activation of the reaction characterized by k and AFB is the free energy of binding substrate to enzyme as determined by K , . If AFT is similar to the free energy of activation of the homogeneous reaction catalyzed by such a group, then the numerical contribution of the free energy of binding is clear. However, the model of enzyme-catalyzed hydrolysis reactions presented here has an additional degree of complication, since the binding of the substrate not only brings the catalytic group of the enzyme (imidazole) into the vicinity of the reactive part of the substrate, but also brings another group of the enzyme into such a position that it forms an acyl compound with the acidic part of the substrate. Studies with model compounds simulating such a situation have not yet gone very far. It may perhaps be wise to await the maximum amount of detail which can be obtained from studies of the enzyme mechanisms before embarking on the difficult task of making suitable models. It is not very surprising that the evaluation of the heats of activation of various enzyme-catalyzed ester hydrolysis reactions has proved to be uninformative, the values being very close to those of base-catalyzed hydrolysis of esters. It may, however, prove very useful when the work on the differentiation between separate steps, described in this paper, can be extended by a study of the effect of temperature on the different steps.
ACKNOWLEDGMENT Much of the work described in this paper was carried out in collaboration with Dr. Julian M. Sturtevant, whose constant advice and help in many ways is gratefully acknowledged.
Received: April 6 , 1966 (in revised f o m June 18, 1956).
29.
ENZYME-CATALYZED HYDROLYSIS REACTIONS
293
REFERENCES I . Foster, R. J., and Niemann, C., Proc. Natl. Acad. Sci. (U.S.) 39, 371 (1956). 2. Wilson, I. B., and Calib, E., J. Am. Chem. SOC.78, 202 (1956). 3. Smith, Emil L., Finkle, B. J., and Stockell, A., Discussions Faraday Soc. NO. 20, 96 (1955). 4. Gutfreund, H., Discussions Faraday SOC.No. 20, 167 (1955). 6. Gibson, Q. H., Discussions Faraday,Soc. No. 17, 137 (1954). 6. Gutfreund, H., and Sturtevant, J. M., Biochem. J . 63, 656 (1956). 7. Chance, B., in “The Mechanism of Enzyme Action” (W. D. McElroy and B. Glass, eds.), p. 399. John Hopkins Press, Baltimore, 1954. 8. Gutfreund, H., and Sturtevant, J. M., Proc. Natl. Acad. Sci. (U.S.)42, 719 (1956). 9. Gutfreund, H., Trans. Faradag SOC.61, 441 (1955). 10. Hammond, B. R., and Gutfreund, H., Biochem. J. 61, 187 (1955). 11. Hartley, B. S., and Kilby, B. A., Biochem. J . 60,672 (1952). fb. Bernhard, S. A., and Gutfreund, H., Biochem. J. 63, 61 (1956). 18. Durell, J., and Fruton, J. S., J. Biol. Chem. 207, 497 (1954). 1.4. Morton, R. K., Nature 172, 65 (1953).