Activation Parameters for the Carbonic Anhydrase 11-catalyzed ...

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Aug 5, 1985 - Ameen F. Ghannam, William Tsen, and Roger S. RowlettS: From the ..... Table I were used. Rate constant". SIVb. SIVIK' k,. 0.0093. 0.8990 k3.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 3, Issue of January 25, pp. 1164-1169,1986 Printed in U.S.A.

Activation Parameters for the Carbonic Anhydrase 11-catalyzed Hydration of C 0 2 * (Received for publication, August 5, 1985)

Ameen F. Ghannam, William Tsen, and RogerS. RowlettS: From the Department of Chemistry, Colgate University, Hamilton. New York 13346

We have determined the activation parameters of kcat and k,,,/K, for the carbonic anhydrase 11-catalyzed hydration of C02. The enthalpy and entropy of activation for kcatis 7860 2 120 cal mol" and -3.99 f 0.42 cal mol" K-', respectively, for the human enzyme. Results for the bovine enzyme were statistically indistinguishable from those of the human enzyme. The entropy of activation of kc,, for the human enzyme was further decomposed into partially compensating electrostatic(es) (AS*, = +15.1 cal mol" IC-') and nonelectrostatic(nes) (AS*nes= -19.1 cal mol" K-') terms. Computer simulations of a formal kinetic mechanism for carbonic anhydrase 11-catalyzed C 0 2 hydration show that 82%of the temperature effect on kc,, can be attributed to the temperature effect on the intramolecular proton transfer step. The reported activation parameters are consistent with a substantial enzyme or active site solvent conformational change in the transition stateof the intramolecular proton transfer step, and is consistent with the mechanism of proton transfer proposed by Venkatasubbanand Silverman (Venkatasubban, K. S., and Silverman, D. N. (1980) Biochemistry 19, 4984-4989).

Carbonic anhydrase is a zinc-containing enzyme that catalyzes the reversible hydration of carbon dioxide (Equation 1). COz

+ Hz0 + HCOT + Hf

implies that the rate-limiting step in the catalysis of CO, hydration must be the intramolecular proton transfer. Computer models have been devised for the kinetic mechanism of carbonic anhydrase I1 based on and expandingupon Steiner's original proposal (Lindskog, 1984; Rowlett, 1984). These computer models have proven consistent with experimentally observed valuesof kcatand K , for the CO, hydration and HCO; dehydration reactions, as well as the known effects of pH, buffer concentration, solvent deuterium content, and inhibition by monovalent anions, phenol, and Cuz+ ion. We have measuredthe temperature and solvent dependence of kc,, and K, for the hydrationof CO, by human andbovine carbonic anhydrase TI a t high pH and non-rate-limiting buffer concentrations. From these measurementswe have estimated entropies and enthalpies of activation for these kinetic parameters for both enzymes. The computermodel of Rowlett (1984) predicts simple correlations between the kinetic parameters kc,, and kc,,,/K, and specific steps in the proposed mechanism, under these experimental conditions. Consequently it is possible to assign the measured activation parameters to discrete steps in themechanism, and in particular theproposed intramolecular proton transfer. The sign and magnitude of the measured activation parameters is consistentwith the idea of a rate-determining intramolecular proton transferbetween a zinc-water complex andhistidine 64 (Venkatasubbanand Silverman, 1980).

(1)

EXPERIMENTALPROCEDURES

Materials-Human carbonic anhydrase was obtained from human erythrocytes of outdated blood by an affinity chromatographyproceThe proton of Equation 1 is transferred from the active site dure (Khalifah et al. 1977). Bovine carbonic anhydrase was obtained to solvent ina complex manner. Steineret al. (1975) proposed fromSigma and was further purifiedviaaffinity chromatography a kinetic mechanism which the proton of Equation 1 is first followed by ion exchange chromatography on DEAE-cellulose. Endeposited onthemetal ion as azinc-water complex, and zyme concentrations were estimated from absorbance measurements subsequently undergoes an intramolecular proton transfer to a t 280 nm by using a molar absorptivity of 5.4 X lo4 liter mol" cm" a neighboring histidine residue. The proton on the histidine for bovine carbonic anhydrase (Pocker andDickerson, 1968) and 5.6 X lo4 liter mol"cm" for human carbonic anhydrase I1 (Coleman, is subsequently transferred in an intermolecular process to 1967). Ches' and thymol blue were obtained from Sigma. Distilled buffer molecules in the bulk solvent (Jonsson et al., 1976; water was passed through two ion-exchange columns prior to use to Silverman and Tu, 1975; Rowlett and Silverman, 1982). At prevent contamination with adventitious metal ions. All other rebuffer concentrations of 10 mM or above, the intermolecular agents were ACS grade or better and used without further purificaproton transfer occurs at a rate too fast to be rate-limiting tion. Kinetic Methods-Saturated solutions ofCO, were prepared by (Silverman and Tu,1975; Rowlett and Silverman, 1982). The bubbling CO, gas into water in a vessel maintained a t 25 "C, and rate of C02/HC0: exchange(exclusive of proton transfer dilutions were prepared in the absence of air by coupling two syringes steps) as measured by isotopeexchange (Silverman et al., as described by Khalifah (1971). Carbon dioxide concentrations were 1979) or NMR line-broadening (Simonsson et al., 1979) is calculated on the basis of 33.8 mM a t 25 "C for a saturated solution at least three times the turnover number for the enzyme. This in water (Pocker andBjorquist, 1977) or in ethyleneglycol solutions, by back-titration with standard barium hydroxide and standard HC1 in the presence of phenolphthalein (Gibbons andEdsall, 1963). Meas*This work was supported by thePetroleumResearchFund, administered by the American Chemical Society; the National Sci- urements of pH and the apparent protonic activity, pH* (Fink and Greeves, 1979), were made with an Orion Model 601A pH ion meter ence Foundation, and Research Corp. The costsof publication of this article were defrayed in part by the payment of page charges. This equipped with a Corning combination glass electrode. Solution exarticle must therefore be hereby marked "aduertisement" in accordThe abbreviation used is: Ches, 2-(cyclohexylamino)ethaneance with 18 U.S.C. Section 1734 solely to indicate this fact. sulfonic acid. $ T o whom correspondence should be addressed.

1164

1165

Anhydrase Carbonic Activation Parameters for posure to the elect,rode was minimized to prevent chloride ion contamination. Initial rates of CO, hydration were measured on a Hi-Tech SF4 stopped flow spectrophotometer equipped with an LTA-403 low temperature control unit and interfaced with an Apple IIe microcomputer/rapid data acquisition system. The flow circuit in this apparatus is entirely Teflon and glass, yielding a gas-tight, solvent-resistant system free of potentially deleterious adventitious metal ions. The observation cell and most of the flow circuit is completely immersed in a thermostatted fluid bath to minimize temperature-induced artifacts. Experiments were carried out with one drive syringe containing the appropriate concentration of COZ in water or solvent, the other containing enzyme, buffer, and indicator dye, in the same solvent. Analysis of Kinetic Data-Estimates of b, and K, from initial rate Versus substrate concentration data were acquired using a nonlinear least-squares method (Cleland, 1.967; Wilkinson, 1961). The initial velocity of hydration (0) was determined according to Equation 2, with dA/dt, the rate of change of absorbance of the indicator, determined by least-squares analysis of absorbance versus time traces comprising less than 10% of the complete reaction. u = -(d[CO~l/dt)initia~ = Qo(dA/dt)inithr

(2)

The buffer factor Qo relates changes in absorbance of the indicator to changes in concentration of H+ion and was determined by titration of buffer-indicator solutions with standard HCI (Khalifah, 1971). Values for A € P and AS* were obtained by fitting rate constant uersw temperature data by a nonlinear least squares method to the Eyring equation,

log k

n 5.8

n

0

8 8 . 8 8 n S 8,&3R

*

8.88335 0.88348 8.88345 0. 88358 8.d355 8 . l/T (I/K>

FIG. 2. Temperature dependence of k,$ (El), K,,, (A), and k,.J K,,, (0)for bovine carbonic anhydrase 11. Calculated activation energies, in kcal mol", are 8.57 +. 0.33 (kcat), 7.39 +. 0.88 (Km), and 1.19 +. 0.70 (kcat/&,). Reaction conditions are as described in Table I. K , (mol liter"} is offset +9 log units; kcaJKm(mol liter" s-') is offset -3 log units.

K,,, calculated from Figs. 1 and 2 are statistically indistinguishable. Values for AH* and AS* for kcatand kat/K,,,,determined by k = (kbT/h)e'S/R- M/Rn (3) fitting the data in Figs. 1 and 2 to the Eyring equation, are where kt, = Boltzmann's constant, h = Planck's constant, R = ideal shown in Table I. For human carbonic anhydrase 11, AH* is gas constant, andT = absolute temperature. Estimates for the electrostatic Contribution to AS* were deter- relatively large and positive for kc,,, and essentially zero for The entropy of activation, AS*, is small for kcst and mined according to Barnard and Laidler (1952), from the variation kC,,JKm. Results for the bovine enzyme of kc, or k.,/K,,, with solvent dielectric constantat 25 "C. The large and negative for kaJKm. dielectric constants for ethylene glycol/water mixtures were obtained are indistinguishable from the human enzyme. from standard tables (Travers and Douzou, 1974). The entropy of activation obtained for the human enzyme Calculation of Sensitiuity Indices-Sensitivity indices were calcu- was further decomposed into the electrostatic and noneleclated according to Ray (1983) using a method of computer simuIation trostatic components using the method of Barnard and Laidof the carbonic anhydrase I1 mechanism previously reported (Rowlett, ler (1952). It is recognized here that such a decomposition of 1984). the activation entropy is only approximate, as factors other than solvent dielectric can influence the rate of reaction in RESlJLTS Arrhenius plots for the kinetic constants kcat,K,, and kcat/ aqueous organic solvents e.8. ionic strength, water concentraK,,, are shown for human (Fig. 1) and bovine (Fig. 2) carbonic tion, specific solvation, and perturbation of acid dissociation anhydrase 11. For both enzymes and all kinetic parameters, constants. However, for carbonic anhydrase 11, minor variations in ionic strength do not appear to have a major effect the Arrheniusplots were linear, suggesting that the ratedetermining step does not change over the temperaturerange on the observed rate of reaction. Ethylene glycol is a very +5 to +30 "C. The activation energies for kcat,K,,,, and kcat/ mildorganic co-solvent for this enzyme, and hasno significant effect on the structure of the enzyme, as measured by intrinsic fluorescence, and no specific solvation/inhibition discernible from extensive studies of the p-nitrophenylacetate hydrolysis reaction.* The contribution by electrostatic effects to the entropy of activation of human carbonic anhydrase I1 was determined from Equations 4 and 5, En k = In k,

(AS*),

- A/T (1 - 1/D)

2 :

(4)

1.13 X 10"' A

15)

where k is the observed rate constant,Ll is the solvent dielectric constant, and (AS"*), is the electrostatic contribution to the activation entropy. Equation 4 describes the dependence 5. of the observed rate constant of the solvent dielectric. The parameter A , obtained from the slope of a plot of In k uersus 4. l/D (Fig. 3 ) , depends on the amount and sign of charges 8. produced in the activated state, and their distance apart, I / T (I/K) according to simple electrostatic theory(Laidler, 1950). EquaFIG. 1. Temperature dependence of &et (a), K m(A), and kWJ K , (0)for human carbonic anhydrase 11. Calculated activation tion 5 relates the parameter A to the electrostatic entropy of energies, in kcal mol-', are 8.58 k 0.18 (kat), 7.38 k 0.89 (K,,,),and activation (Barnard andLaidler, 1952). For human carbonic anhydrase 11, we find that there is a 1.20 +. 0.82 (kcat/&). Reaction conditions are as described in Table 5.

I. Km (mol liter-') is offset +9 log units; Lt/r;i,,, (mol liter" s-l) is offset -3 log units.

-

* R. S. Rowlett, unpublished observations.

-

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Activation Parameters for CarbonicAnhydrase TABLEI Activation parameters for the carbonic anhydrase ZZ-catalyzed hydration of CO, The following conditions were used: pH 9.0; 10 mM Ches; 25 p~ thymol blue; ionic strength, 0.20 (Na2S04). Human

AH* (cal mol") A S * (cal mol" K-I)

In k

Bovine

kcat

kcatlKm

kcat

k d K m

+7860 f 124 -3.99 0.42

+604 t 808 -18.7 f 3.0

+8144 f 445 -3.54 f 1.50

+520 f 716 -19.9 f 2.5

3 I

I

SCHEME1. Formal kinetic mechanism for carbonic anhydrase I1 catalysis of the Con.The various species involved in the catalytic mechanism are denoted asfollows: E , enzyme; H, hydrogen I1 ion; BH, protonated buffer; B , unprotonated buffer; S, carbon dioxide; 8.8128 e . 8 h e.8'13 8 . e i n 8.8'134 e . 8 ' 1 ~ e.ei3a 8.8148 and P, bicarbonate ion. X denotes a transient intermediate between 1/D CO, and HCO;. H to the left of E denotes protonation a t His-64. H FIG. 3. Solvent dielectric dependence of kc,, (0) and kCat/Km to the right of E denotes protonation of the zinc-hydroxide group. (A) for human carbonic anhydrase 11. Reaction conditions are as The values of the rate constantsin Scheme 1 are described elsewhere (Rowlett, 1984). described in Table 11. 12

I

experimental conditions the intermolecular proton transfer is diffusion-limited, and hence can exhibit little or no solvent deuterium isotope effect (Rowlett and Silverman, 1982). Additional, indirect evidence for a reaction scheme which includes a rate-determining intramolecular proton transfer has been provided by computer simulations of the Steiner mechkcat k d K m anismanditsvariations(Lindskog, 1984; Rowlett,1984). -7.84 f 3.13 +15.1 f 2.9 AS*,, These computer models are able to accurately and quantita-10.9 f 6.1 As*nee -19.1 3.3 tively predict the pH dependence and magnitude of kc,, and K,, account for the effect of external buffer concentration on sizable positive electrostatic entropy of activation for kcatand the observed turnover rate, and reproduce the complex pHa slightly negative electrostatic entropy of activation for kcat/ dependent behavior of inhibition caused by monovalent anK,. The non-electrostatic contributions to the activation en- ions, phenol,or Cu2+ion. Scheme 1is the simplestmodel that tropies, (AS*),,,, have been calculated from Equation 6, and is consistent with all of the extant kinetic data (Rowlett, 1984). are shown in Table 11. According to Scheme 1, the rate-limiting step, defined here AS* = (AS*),, (AS*),,, (6) as the step following the intermediate which accumulates to the greatestdegree in steady state, changeswith pH, and toa lesser degree, with CO, concentration (Rowlett, 1984). At low DISCUSSION pH values, where the rate of proton transferbetween enzyme The kinetic model of carbonic anhydrase I1 catalysis origi- and external buffer is inefficient according to the Eigen relanally proposed by Steiner et al. (1975) suggests that the proton tionship (Rowlett and Silverman, 1982), the intermolecular produced in the reaction of Equation 1 is transferred tobulk proton transfer is rate-limiting; that is, the rate of turnover solvent in two distinct steps. The first, and rate-limiting stepis limited by k7[B]in Scheme 1. At high pH, where intermolecular protontransfer is nearlydiffusioncontrolled, the undermostexperimentalconditions,isanintramolecular rate-limiting: the rate of proton transfer from a zinc-water complex to a nearby accep- intramolecular proton transfer is tor group on the enzyme, probably His-64. This proton sub- turnover is limited by k5. However,solvent deuterium isotope and temperature efsequently,in asecond step, undergoes anintermolecular transfer tobulk solvent, where buffer molecules appear to the fects on enzyme-catalyzed reactions are more appropriately actual acceptor species (Silverman and Tu, 1975; Jonsson et considered by using the most sensitive step of the kinetic al., 1976; Rowlett and Silverman, 1982). The primary evidence mechanism (Ray, 1983; Northrop, 1983). The most sensitive a step is defined as that step, which, when changed, has the for a rate-determining intramolecular proton transfer is residual solvent deuteriumisotope effect on kcatof 3-4 at high largest influence on one of the measured kinetic rate constants. A quantitative measure of thissensitivitycan be pH in the presence of high concentrations of buffer species (Steiner et al., 1975; Pocker and Bjorquic-t 1977). ITnder these afforded by calculating a sensitivity index for each individual TABLE I1 Estimates of electrostatic and nonelectrostatic entropies of activation for the human carbonic anhydrase ZZ catalyzed-hydration of CO, The following conditions were used: pH* 9.0; 10 mM Ches; 100 PM thymol blue; ethylene glycol was used as co-solvent.

*

+

Anhydrase Carbonic Activation Parameters for

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enzyme would still be 90% in the active form. A reasonable estimate of the possible temperature-induced perturbation of the pK, of this group on going from +25 to +5 "C is between 0.8 and 1.0 pH units, given an enthalpy of ionization of 7-9 kcal/mol, reasonable values for a metal-bound water or imiRate dazolium species (Cohn and Edsall, 1943). Thus, under our SIVb SIVIK' constant" experimental conditions, the ionization state of the enzyme 0.8990 k, 0.0093 is changed only minimally.Another considerationis the effect 0.0902 k3 0.0091 of temperature on the buffer-indicator system that is used to 0.0090 ks 0.8216 monitor the progress of the reaction. Wehave addressed this 0.0018 k7 0.1600 by measuring the buffer factor, Qo, directly at each temperaRate constant of Scheme 1. ture using standardizedHC1. * Sensitivity index for maximal velocity or kcat. Two previous studies of the temperature dependence of Sensitivity index for the ratio of maximal velocity to the Michaelis carbonic anhydrase I1 catalysis have been reported, but are constant, or kCat/K,. notinagreement.SanyalandMaren (1981) reportedan activation energy of 8.15 kcal/mol on kc,, for human carbonic step in the mechanism (Ray, 1983). The sensitivity index for anhydrase 11, similar to our value. However,Pocker et al. a reactionsteprepresentstheproportion of the observed (1981) reported an activationenergy of 5.01 kcal/mol for the change in the experimentally monitored kinetic constant that kinetically identical bovine enzyme. We do not agree with is contributedby that reaction step3 either study on the temperature effect on K , for the CO, In Scheme 1,the most sensitive stepwith respect to kc,, for hydration reaction. Sanyal and Maren (1981) could not detect the CO, hydration (forward) reaction at all p H values and a temperature effect on K,,, for the human enzyme, but their experimentally reasonablebuffer concentrations, is the intra- method did not yield precise enough values ofK,,, to make a molecular proton transfer: kcat is limited by kg in Scheme 1. valid judgment of this question. Pockeret al. (1981) reported The sensitivity index for this step calculated from Scheme 1 a very small if anytemperature effect on K , for bovine using the experimental conditions describedin this paper, carbonic anhydrase. We are unable to explain these differpredicts that the temperatureeffect upon the intramolecular ences, butnotethatSanyalandMarencarriedouttheir proton transfer step contributes82% of the observed temper- experiments at pH 7.5, and Pocker et al. (1981) at pH 8.5, ature effect on kc,, (Table 111). The most sensitive stepin the while our experiments were done at pH 9.0 for reasons outCO, hydration reaction with respect to the kinetic constant lined previously. k,,,/K,,, is the first step, in which CO, combineswith the Although the formal kinetic mechanism of carbonic anhyenzyme and is transformed into bicarbonate: k,,,/K,,, is condrase I1 catalysisrepresented by Scheme 1 seems to adetrolled by kl[S] in Scheme 1. The sensitivity index calculated quately describe theexperimental kineticbehavior of the for this step predicts that the observed temperature effect on enzyme (Rowlett, 1984; Lindskog, 1984), little is known of the h,,,/K,,, is 90% derivedfrom the temperature effect on the chemical aspects of the individual steps in the mechanism, rate of that step (Table11). Thus, it seems that the computer models allow a correlation between temperature effects on especially the assumed intramolecular proton transfer step, steady-state kinetic parameters and elementary stepsin car- designated ks in Scheme 1. That this stepis a proton transfer is assumedfrom the residual solvent deuterium isotope effect bonic anhydrase I1 catalysis.Specifically, thetemperature buffer concentrations of 3-4 on kc,, at high pH and saturating effect on h,, offers insightinto k5 of Scheme1,andthe (Steiner et al., 1975; Pocker and Bjorquist, 1977). Venkatatemperature effect on kCat/K,,,offers insight into k l of Scheme subban and Silverman (1980) further explored the solvent 1. Despite such correlations, the treatment and interpretation deuterium isotope effect on kc,, a t varying mole fractions of of temperature effects upon enzyme-catalyzedreactions is not deuterium. They concluded that there were more than one, and probably three protonsin flightduring the rate-determintrivial,andthereareotherconsiderations which mustbe satisfactorilyaddressed.Most troublesome is the effect of ing (intramolecular proton transfer) step. These protons are temperature on the ionization state of the enzyme. Temper- postulated to be contributed by two water molecules which ature will have aneffect on thepK, values of ionizable groups form a bridge between the zinc-water complex and the posin the active siteresponsible for catalysis andpossibly change tulated acceptor, His-64. This hypothesis is reasonable covthe concentration of the active form of the enzyme. Experi- sidering that protondonor and acceptor are separatedby 6 A; mental conditions must be chosen to minimize this effect, if the zinc ion in carbonic anhydrase I1 lie: at the bottom of a possible. The activity of carbonic anhydrase I1 appears to be conical cavity in the enzyme nearly 15 A deep, while His-64 controlled by a single ionizing group whose pK, is near 7.0; lies near the surface of the enzyme, in the samecavity (Kanthis group must be in the basic form for the enzyme to be nan et al., 1977). Is such a hypothesis of a rate-determining intramolecular active. Under our experimental conditions (pH 9.0), the enproton transfer consistent with the observed activation pazyme is 99% in the active form at 25 "C. If the pKa of this group were to be perturbed upwards by one whole pH unit at rameters? One normally considers proton transfer reactions in water between oxygen/nitrogen donor/acceptors to be very 5 "C, the lowest temperature used in our experiments, the rapid,with low activation energies. For example, the rate The normalized sensitivity index for a reaction step is quantita- constant of proton transferbetween water molecules in dilute tively defined as [d(l/c)/d(l/k,)][c/ki], where c is the measured kinetic buffer solutions is near 10"' M" s" at room temperature, with constant (e.g. kcat or kCat/Km), and k: is the rate constant for the ith an activation energy of 3-5 kcal/mol, depending on the pH step in the forward reaction. The sum of the sensitivity indices for (Loewenstein and Szoke, 1962). Likewise, the rate of proton the i steps in the forward reaction is exactly 1. Thus, the sensitivity indices relate the efficiency with which perturbations in a single rate transfer between methylammonium ion andits conjugate constant (caused by temperature, isotope effects, etc.) are reflected in base, either directIy or mediated by a single water molecule is 10" M" s" at room a measured kinetic constant. For a more complete treatment, see Ray very fast, witha rateconstantnear (1983). temperature, and an activation energy near zero (Connor and TABLEI11 Calculated sensitivity indices forthe carbonic anhydrase II-catalyzed hydration of COn The method of Rowlett (1984) and the experimental conditionsof Table I were used.

1168

Activation Parameters for

Carbonic Anhydrase

Loewenstein, 1961). Similar results are observed for the di- for k,,,/Km is alsolarge and negative, consistent with the methyl-andtrimethylammoniumions(Grunwald, 1963); formation of the negatively charged HCO; from the neutral Loewenstein, 1963). For carbonic anhydrase 11, the intramo- co,. It is difficult to satisfactorily explain at this time the reason lecular proton transfer stepk5 is quiteslow, about lo6 M-’s-’, and its activation energy is quite large, near 8.6 kcal/mol; as why there has tobe an intramolecular proton transfer atall, estimated from the temperature dependence of kc,,. although the existence of such a step is strongly inferred by Alwyn Jones, Wallenberg Laboratory, Uppsala, recently has kinetic studies (Steiner et al., 1976; Pocker and Deits, 1982), refined the crystal structureof human carbonic anhydrase I1 solvent deuterium isotope effects (Steiner et al., 1975), and ordered water computer simulations (Lindskog, 1984; Rowlett, 1984). Why (Lindskog et al., 1984) anditindicatesan structure in the active site, and ahydrogen bond between cannot buffer molecules, the normal acceptors for the proton Thr-199 and the zinc-bound water molecule. His-64, the ac- generated in Equaton 1, interactdirectly with the zinc-bound ceptorofor the proton transfer from the zinc-water complex, water molecule? The possibility of steric inaccessibility has is 3.3 A away from the closest water molecule in the ordered been discarded on the grounds there are a number of large structure. Thus, a rapid, diffusion controlled Grotthus mech- inhibitor molecules, such as aromatic sulfonamides, phenols, anism of proton transfer may be impeded by a discontinuity and thiophenols, which are known to interact directly with of the water chain between His-64 and the zinc ion. The themetal ion via acid/basesites(Lindskog et al. 1984). estimated entropy of activation for kc,, (the intramolecular However, it should be noted that all of the above-mentioned proton transfer step) is very nearly zero. The simplest inter- inhibitor molecules are significantly nonpolar in nature, and pretation of this result is that there isvery little reorganiza- all presumably interact with the nonpolar portion of the active tion of either solvent or of the enzyme in the transition state site (Jacob et al., 1980). Buffer molecules such as the comof this proton transfer. However, an alternative interpretation monlyused “ G o o d buffers(Good, 1966) are considerably is that there are very nearly compensating electrostatic and more polar than these aromatic inhibitors. One might specunonelectrostatic entropies of activation. Using the method of late that these buffer molecules cannot favorably interact Barnard and Laidler (1952) we have estimated the electro- with the nonpolar region in the active site, and are thus poor static and nonelectrostatic contributions to the entropy of agents for direct proton transfer from the zinc-bound water activation for the intramolecular proton transfer step. The molecule. electrostaticcomponent of theactivationentropycan be We conclude that the activation parameters measured for viewed as being derived primarily from charge formation or the human and bovine forms of carbonic anhydrase I1 are separation in the transition state. The nonelectrostatic con- consistent with the formal kinetic mechanism of Scheme 1 tribution of the activation entropy can be regarded as being and the notion of a rate-determining intramolecular proton derived from enzyme conformational changes, nonelectro- transfer.Theapplication of sensitivity indices derived by strictive solvent reorganization, or simple bimolecular inter- calculation fromScheme 1allows the assignmentof activation action. Our results (Table 11) seem to imply that there is a energies, measured for steady-state kinetic constants, to spedecrease inchargeconcentration or separation during the cific elementary steps in the formal kinetic mechanism. The proton transfer (a positive electrostatic entropyof activation), enthalpy and non-electrostatic entropy of activation for the and a substantial reorganizationof enzyme conformation and/ intramolecularprotontransferstep is consistent with an or possibly solvent (a large negative nonelectrostatic entropy enzyme conformational change prior to or during the actual of activation). This result seems consistent with the proton transfer, and could account for the anomalously slow rate for transfer mechanism proposed by Venkatasubban and Silver- proton transfer between oxygen and nitrogen. man (1980) and the active site proposed by Lindskog et al. REFERENCES (1984). If we assume His-64 is not in contact with the ordered waterchain/zincionintheactivesite,then a structural Barnard, M. L., and Laidler, K. J . (1952) J . Am. Chem.Soc. 7 4 , 6099-6101 reorganization would be necessary before protontransfer Cleland, W. W. (1967) Adu. Enzymol. Relut. Areas Mol. Biol. 29, 1could take place. This enzyme/solvent reorganization could 32 account for the negative nonelectrostatic entropyof activation Cohn, E. J. and Edsall, J. T. (1943) Proteins,AminoAcids,and for the intramolecular proton transfer step. However, once Peptides, Van Nostrand Reinhold Co., New York His-64 is brought into contact with the zinc ion through the Coleman, J . E. (1967) J. Biol. Chem. 242, 5212-5219 extended water chain, proton transfer could occur through a Connor, T. M., and Loewenstein, A. (1961) J. Am. Chem. Soc. 8 3 , 560-563 Grotthus-like mechanism. The spreading outof the chargeof the transferred proton throughout the water chain could then Fink, A. L., and Geeves, M. A. (1979) Methods Enzymol. 6 3 , 336370 account for the positive electrostatic entropy of activation. The activation parameters obtained for kCat/Km can be used Gibbons, B. H., and Edsall, J. T. (1963) J. Biol. Chem. 238, 35023507 as a check of the validity of the results obtained fork,,,, and Good, N. E. (1966) Biochemistry 5 , 466-477 of the utility of Scheme 1 as well. According to Table 111, Grunwald, E. (1963) J. Phys. Chem. 67,2208-2214 are essentially attributable Jacob, G. S., Brown, R. D., 111, and Koenig, S. H. (1980) Biochemistry activation parameters for kCat/Km 19,3754-3765 to the step kl[S] in Scheme 1. This is the step in which C 0 2 binds to theenzyme and is converted intoHCO; and isfairly Jonsson, B. H., Steiner, H., and Lindskog, S. (1976) FEBS Lett. 6 4 , 310-314 well understood compared to the intramolecular proton transKannan, K. K., Vaara, I., and Notstrand, S. (1977) in Drug Action at fer step. Quantum mechanical calculations of the attack of the Molecular Leuel, (Roberts, G. C. K., ed) p. 73, University Park zinc-boundhydroxide on carbon dioxide indicate that this Press, Baltimore process has little or no activation energy (Pullman, 1981). Khalifah, R. G. (1971) J. Biol. Chem. 2 4 6 , 2561-2573 Correspondingly, the activation energy or enthalpy of acti- Khalifah, R. G., Strader, D. J., Bryant, S. H., and Gibson, S. M. (1977) Biochemistry 16, 2241-2247 vation of k,,,/K, is very small, andprobably indistinguishable Laidler, K. J. (1950) Chemical Kinetics, p. 133, McGraw-Hill, New from zero. However, the entropy of activation for k,,,/K,,, is York large and negative, which is consistent with abimolecular Lindskog, S. (1984) J . Mol. Catal. 23, 357-368 process. In addition, the electrostatic entropy of activation Lindskog, S., Engberg, P., Forsman, C., Ibrahaim, S. A., Jonsson, B.-

Activation Parameters H., Simonsson, I., and Tibell, L. (1984)Ann. N.Y. Acad. Sci. 429, 61-75 Loewenstein, A. (1963)J. Phys. Chem. 67, 1728-1730 Loewenstein, A., and Szoke, A. (1962)J. Am. Chem. SOC.84, 11511164 Northrop, D. B. (1981)Biochemistry 20,4056-4061 Pocker, Y., Deits, T. L., andTanaka, N. (1981) in Aduances in Solution Chemistry (Bertini, I., Lunazzi, L., and Dei, A., eds) pp. 253-274,Plenum, New York Pocker, Y., and Bjorquist, D. W. (1977)Biochemistry 16,5698-5707 Pocker, Y., and Deits, T. L. (1982)J. Am. Chern. SOC.104, 24242434 Pocker, Y., and Dickerson, D. G . (1968)Biochemistry 7,1995-2004 Pullman, A. (1981)Ann. N . Y. Acad. Sci. 367,340-355 Ray, W. R., Jr. (1983)Biochemistry 22,4625-4637 Rowlett, R. S. (1984)J. Prot. Chem. 3,369-393

for Carbonic Anhydrase

1169

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