THE OF BIOLOGICAL Vol. 266, No. 5, pp. 20636-20644.1991 Q ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1991 by The American Society for Biochemistry and Molecular Biology, Inc Vol.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. Issue of November 5, pp. 20636-20644.1991 Printed in U.S.A.

The pH Variation of Steady-state Kinetic Parameters of Site-specific Co2+-reconstituted Liver Alcohol Dehydrogenase A MECHANISTIC PROBE FOR THE ASSIGNMENT OF METAL-LINKED IONIZATIONS*

(Received for publication, March 12, 1991)

Wolfgang MaretS and Marvin W. Makinen5 From the Department of Biochemistry and Molecular Biobgy, The University of Chicago, Cummings Life Science Center, Chicago, Illinois 60637

To identify ionizations of the active site metal-bound water in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1) , the pH, solvent isotope, temperature, and anion dependences of the steady-state kinetic parameters kcat and koat/KM have been evaluated under initial velocity conditions for the native and the active site-specific Co’+-reconstituted enzyme. In the oxidation of benzyl alcohol, a bell- shaped pattern of four prototropic equilibria was observed under conditions of saturating concentrations of NAD+. It is shown that the ionizations governing kcat (pK1 = 6.7, pK2 = 10.6) belong to the ternary enzyme- NAD+-alcohol complex, whereas the ionizations governing k,.JKM (pK1’ = 7.5, pKz’ 8.9) belong to the binary enzyme-NAD+ complex. The ionizations pK1 and pK1‘ are not influenced by metal substitution and are ascribed to His-61 on the basis of experimental estimates of their associated enthalpies of ionization. On the other hand, pK2 and pK2‘ are significantly decreased (ApK,, = 1.0) in the Co2+-enzyme and are attributed to the active site metal-bound water molecule. The shape of the pH profiles requires that the metal ion coordinates a neutral water molecule in the ternary enzyme-NAD+-alcohol complex under physiological conditions. The possible catalytic role of the water molecule within a pentacoordinate metal ion complex in the active site is discussed.

The mechanism of action of liver alcohol dehydrogenase (LADH,’ alcohol:NAD+ oxidoreductase, EC 1.1.1.1) centers on different ligand states of the active site Zn2+ ion during the catalytic cycle (2), conformational changes in the protein (3-5) that govern the affinity and kinetics of coenzyme binding (6,7), and temporal separation of proton abstraction from

* This work was supported by Grant AA 06374 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$Present address: Center for Biochemical and Biophysical Sci- ences and Medicine, Harvard Medical School, Boston, MA 02115.

3 To whom correspondence should be sent: Dept. of Biochemistry and Molecular Biology, The University of Chicago, Cummings Life Science Center, 920 East 58th St., Chicago, IL 60637.

’ The abbreviations used are: LADH, (horse) liver alcohol dehydrogenase; ZnLADH, the native, Zn2+-containing enzyme; CoLADH, active site-specific Coz+-reconstituted LADH as described in Ref. 1; BzOH, benzyl alcohol; Caps, 3-(cyclohexylamino)-l-propanesulfonic acid; Ches, 2-(N-cyclohexylamino)ethanesulfonic acid; Hepes, N-2- hydroxyethylpiperazine-N‘-2-ethanesulfonic acid; Pipes, 1,4-pipera- zinediethanesulfonic acid; TES, N-tris(hydroxymethy1)methyl-2- aminoethanesulfonic acid; TFE, 2,2,2-trifluoroethanol.

the chemical step of hydride transfer (7-11). These molecular events are controlled by ionizing groups for which assignment has remained conjectural despite numerous structural, chemical, and kinetic studies. The ionization with pKa x 7 that controls turnover has been attributed to the Zn2+-bound alcohol in a tetracoordinate species (8-10) and to His-51 (11) or to a Zn2+-bound water molecule (5,12) in a pentacoordinate species. Moreover, two ionizations with pKa 9.2 in the free enzyme and pK, = 7.6 in the binary complex formed with NAD+ have been attributed to the metal-bound water molecule (10,13-16). However, it has been demonstrated that the ionization with pK, = 7.6 is unchanged by removal of the active site metal ion (17, 18), strongly indicating that an amino acid side chain is responsible for the observed pH effects. Accordingly, it has been suggested that Lys-228 is responsible for the ionization with pKa = 9.2 in the free enzyme (19). There is, thus, no agreement on basic features of the mechanism of action of LADH, such as assignment of ionizations of the metal-bound water or the alcohol molecule, of groups on the enzyme responsible for acid-base catalysis, or of the coordination number of the active site metal ion.

A direct resolution of many of the ambiguities concerning the molecular origins of pK, values can be achieved by iden- tifying the ionizations of metal-bound ligands. In particular, metal substitution provides an experimental test for metal- sensitive ionizations. For instance, substitution of the open- shell Co2+ for the closed-shell Zn2+ may be expected to result in a detectable decrease in the pKa of a metal-bound ligand. In this respect the active site-specific Co2+-reconstituted enzyme characterized by Zeppezauer and co-workers (1) provides a particularly incisive derivative of LADH because it has been demonstrated that the structure of the enzyme and especially of the active site metal ion region are essentially identical with the native enzyme (20). Ionizations of molecules that reversibly ligate the metal ion can be further probed by subtituting deuterium oxide for natural abundance water or by varying the structure of alcoholic substrates since their ionization constants cover a range of iO”4-iO”9 M in magnitude (21).

In a preliminary study of the influence of the metal ion on ionizations affecting catalysis of LADH, we observed that an ionization with pK. - 10.6 controlling kat and an ionization with pK, - 8.9 controlling kCat,KM were decreased by substitution of Co2+ for the active site Zn2+ (22). To characterize these ionizations in more detail, we have carried out a com- parative investigation of the pH, solvent isotope, substrate, and temperature dependences of the steady-state kinetic parameters that govern the oxidation of alcohols catalyzed by both native LADH and the active site-specific Co2+-reconstituted enzyme. Our analysis shows that these two metal-

20636

Mechanism of Liver Alcohol Dehydrogenase 20637

dependent ionizations are best ascribed to metal-bound water in the ternary enzyme-NAD+-alcohol complex and the binary enzyme-NAD+ complex, an assignment that differs from pro- posals made by others (10, 15, 16, 19). In particular, our results specify that the active site metal ion in the catalytically competent ternary complex is ligated by a neutral water molecule in the physiological pH range. Together with the expectation that the alcohol substrate is also metal bound, as in ternary inhibitor complexes (15,23-25), the results require that the catalytically active metal ion is pentacoordinate. We indicate how the metal-bound water molecule may play a central role i n the reaction mechanism in proton abstraction in light of structural relationships of a putative proton relay system in the active site cleft (15, 23).

EXPERIMENTAL PROCEDURES

Muteriak-NAD+ and NADH (grade 1) were obtained from Boeh- ringer Mannheim. Coenzyme purity was found by enzymatic assay (26) to be 94 and 84%, respectively. 2-Propanol (spectroscopic grade) and TFE (Gold Label) were obtained from Aldrich; Tes, Pipes, Hepes, Caps, and deuterium oxide (99.8% DzO) from Sigma; Ches from Calbiochem; and Tris (Ultra-pure) from Schwarz/Mann. Analytical reagent grade BzOH was distilled under reduced pressure. All other chemicals were of analytical reagent grade. Deionized, distilled water was used throughout.

Crystalline ZnLADH packed for transit a t 0 “C was obtained from Boehringer-Mannheim and used without further purification. Amor- phous and flocculent protein precipitate was discarded. The remain- ing crystalline enzyme was then dissolved, exhaustively dialyzed against 50 mM Tris-HC1 at pH 8.1 and recrystallized by stepwise addition of multiply distilled tert-butanol to the dialysate. The active site specific Co’+-reconstituted form of LADH was prepared and characterized according to Maret et ul. (1). All preparations of Co- LADH corresponded to no less than 85% active site metal ion substitution (1.7 g-atoms of Co2+/dimer). Stock solutions of CoLADH in 25 mM Tes at pH 7, prepared and kept under a nitrogen atmos- phere, showed no change in activity for a t least 1 week. Incubation of CoLADH for up to 15 min in the reaction mixtures used for kinetic studies in the pH range 7-11 resulted in no detectable loss of catalytic activity.

Methods-Initial velocity data were collected with a Perkin-Elmer MPF-44A spectrofluorimeter by following NADH fluorescence at 460 nm with excitation at 340 nm. The increase in emission intensity was calibrated under identical conditions of instrument gain and signal amplitude with solutions of NADH of which the concentration was enzymatically determined (26). The reaction was initiated by addition of enzyme. Initial velocity data used to estimate kinetic parameters were the means of a t least two measurements each; if the deviation of these two measurements was more than lo%, additional data points were collected.

Initial velocity data were collected under conditions corresponding to the steady-state approximation and with saturating levels of coenzyme. Below pH 6, the NAD+ concentration was 25.75 mM, and in solutions of pH >6, the concentration was 21.15 mM, as specified by the pH dependence of the equilibrium dissociation constant of the enzyme-NAD+ complex (27). These concentrations were verified by experiment to provide saturating levels of the coenzyme for both ZnLADH and CoLADH over the entire pH range studied. For reaction mixtures below pH 6, the buffer was readjusted to the required pH value following the addition of NAD+. Above pH 10, NAD+ was added only after temperature equilibration had been achieved in order to minimize decomposition of the coenzyme at high pH (28). The pH of reaction mixtures was found to remain constant to within k0.05 pH units after completion of the reaction in all cases. During collec- tion of initial velocity data, the temperature of the reaction mixture was maintained at 21 k 1 ‘C.

Initial velocity data were evaluated with use of the algorithm ENZKIN provided by Prof. J. L. Westley of the Department of Biochemistry and Molecular Biology at The University of Chicago. The kinetic parameters kat and K, were calculated with this algorithm by an iterative, nonlinear, least squares fit to a rectangular, hyperbolic function. From the pH variation of these kinetic parameters plotted in double-logarithmic form (29), ionization constants were estimated with use of the algorithm BELL (30). For closely overlapping ionizations (ApK, 5 l), the pK, values were estimated by

visual fit of the data to theoretical curves calculated for a single ionization since such closely overlapping ionizations cannot be uniquely analyzed by computer algorithms (30). In general, substrate concentrations were varied over the range of 0.1-2 X KM. Particular attention was paid to avoid conditions of substrate inhibition.

On the basis of initial velocity data, we confirmed that TFE behaves as a linear, competitive inhibitor for both ZnLADH and CoLADH over the entire pH range investigated. Equilibrium dissociation constants of the competitive inhibitor TFE were then determined as a function of pH by a full kinetic analysis a t four inhibitor concentrations in the presence of zwitterionic buffers. The Kr values were estimated on the basis of a least squares fit of the calculated slope of the double-reciprocal plot uersus the inhibitor concentration. For solutions of pH > 8, we observed that incubation of the enzyme in the presence of NAD+ and TFE led to slow formation of an inactivated complex detectable by an increase in fluorescence emission intensity at 460 nm. This species must represent a form of the enzyme with altered kinetic properties since slower rates were observed upon subsequent substrate addition than for initiation of the reaction by the addition of enzyme. For this reason, initial velocity data for inhibition studies were collected only under conditions of initiating the reaction by addition of the enzyme to the buffered mixture of NAD+, TFE, and substrate.

For kinetic studies in DzO, the pD was adjusted with NaOD or DCl according to the relationship pD = pH + 0.4, where pH is the nominal pH reading obtained with the pH meter equipped with a pH glass electrode. For these measurements, all solutions were prepared with DzO, except for the enzyme, and the reaction mixture contained no less than 98% DzO.

RESULTS

Influence of Metal Ion on Ionizations Governing Substrate Oxidation

Oxidation of Benzyl Alcohol and 2-Propanol-No detailed evaluation of the LADH-catalzyed oxidation of BzOH or of 2-propanol has been reported previously on the basis of steady-state kinetic methods. We have chosen BzOH and 2- propanol as substrates in this study because (i) BzOH has been used extensively in transient state kinetic studies (8- IO), (ii) the rate-limiting step with 2-propanol is well established as hydride transfer (31), and (iii) the pK. values of these alcohols vary between 17 and 20 (21). Moreover, the use of these substrates is not influenced by factors that complicate the interpretation of kinetic results. For instance, the oxidation of ethanol is sensitive to substrate inhibition (32, 33), and the oxidation of cyclohexanol is subject to substrate activation or substrate inhibition, depending upon the concentration of NAD’ (11,34). On the other hand, the oxidation of 2-propanol near neutral pH is not subject to substrate inhibition within the range of substrate concentrations used (35), and in this investigation we demonstrated that saturating concentrations of BzOH can be reached without deviation of initial velocities from a hyperbolic relationship.

The influence of pH on the kinetic parameters kat and kcat/ KM in the oxidation of BzOH is compared in Fig. 1 for ZnLADH and CoLADH. The bell-shaped profiles define four prototropic equilibria. The ionizations pK, and pK2 are observed in the pH profile of kcat, whereas pKl’ and pKz’ are observed in the pH profile of kcat/K~. Although the profiles are similar for the two enzymes, the limiting values of kcat and kcat/& are lower for CoLADH than for the native enzyme in the high pH range. The decrease i n the limiting value of kcat/ KM for CoLADH results from the lower plateau value of kcat.

T h e acidic portion of the pH profiles of log kat shows a distinct ionization with pK, 6.2-6.5 for both metalloenzymes. On the other hand, the alkaline portion of the p H profile of log kat for CoLADH shows a hollow accompanied by an approach to a plateau in the pH profile of log kat/&. These two observations specify an ionization in the alkaline pH range that governs kat (36), i.e. the dashed arrows in the

20638 Mechanism of Liver Alcohol Dehydrogenase

I O -

01-

10’-

10‘-

Id -

5 6 7 8 9 1 0 1 1 pH

FIG. 1. Comparison of the pH dependence of the kinetic parameters kt and k J K M for the oxidation of BzOH in the presence of added chloride. In the pH profile of log kat&, the broken arrow indicates the value of the ionization constant governing kc,, as estimated on the basis of the pH profile of -log KM and identified by the approach to a plateau in the pH profile of kat/KM (36) (see text). The other ionization constants are indicated by arrows (J for ZnLADH and t for CoLADH). The square brackets are drawn to indicate the standard error associated with the estimation of each parameter when results for both enzymes are closely overlapping. The following buffers were used at a total concentration of 0.1 M in the presence of 0.1 M potassium chloride: phosphate at pH 5-7, pyrophosphate at pH 7.5-9, and glycinate at pH 9.5-11. In these studies, the enzyme concentration was approximately lo-’ M in active sites.

pH profiles of kat/KM actually designate ionizations that control kmt. The pH profile of log kaJKM for both metalloenzymes is essentially identical on the acidic side. On the alkaline side, however, the profile for CoLADH is similar in shape to that of ZnLADH but is markedly shifted to lower pH values.

Since the approach to a plateau in the pH profile of log kcat/& does not define the value of the ionization constant (cf. Ref. 36, p. 205)) we have also analyzed the pH profiles of -log KM for both ZnLADH and CoLADH (data not shown), for ionizations influencing kat and kat/KM may be observed in the pH profile of KM. The ionization governing log kat with pKa - 6.5 and the two ionizations governing log kat/KM were directly confirmed. The fourth ionization identified in Fig. 1 only as a hollow in the pH profile of kat was directly revealed with an unambiguous metal dependence. The order of the magnitude of the ionization constants pKl < pKl’ < pK2’ < pK2 was the same for both metal-enzymes. In the plot of -log KM versus pH, the ionizations designated as pKl and pKl’ were not measurably influenced by Co2+ substitution, whereas there was a clearly identifiable metal dependence of the values of pK2 and pK2‘.

We have determined similarly the pH dependence of kinetic parameters governing the oxidation of 2-propanol and have compared the values of the ionization constants in Table I to those observed for oxidation of BzOH. Although comparable

values for pK1 and pK1‘ were obtained, no decrease from the limiting plateau value in the pH profile of kat/& up to pH 11 was observed for the oxidation of 2-propanol catalyzed by ZnLADH. In contrast, for CoLADH the onset of the ionization of pK2’ was observed yielding a value of -10.2. A metal- dependent shift similar in magnitude to the one observed for BzOH places this ionization at pK, values 2 11.5 for ZnLADH and, hence, outside of the range of detection by steady-state methods. Similarly, we conclude that the ionization pK2 governing kat in the oxidation of 2-propanol must be shifted also to values >11.5 since the onset of pK2 was observed for CoLADH near pH 11. Thus, in the oxidation of 2-propanol, the ionizing groups governing kat and kcat/KM at high pH are altered by metal substitution, whereas the values of pKl and pKl’ are insensitive to the metal, corroborating the results with BzOH.

Estimates of the ionization constants governing the kinetic parameters for BzOH and 2-propanol oxidation are summarized in Table I for the various reaction conditions employed in this investigation and are compared with corresponding values obtained in studies with other substrates (9,11,13,33, 34,39). We have also included in this table the pKa values of ionizations governing the kinetic parameter d2‘ as derived for ethanol oxidation (40) and applied for steady-state measurements of the oxidation of BzOH (9). The parameter &’ corresponds to the reciprocal of kCat/KM, and the associated pK. values are in not unreasonable agreement with ours.

A variety of anion binding effects on kinetic parameters governing substrate oxidation have been previously described by others (33, 41). In Fig. 2 we have compared the pH dependence of the kinetic parameters kat and kat/& for the ZnLADH catalyzed oxidation of BzOH in the presence and absence of added chloride, as a characteristic anion influencing kinetic parameters. As seen in Fig. 2, the ionization constants governing kcat/& are shifted to lower values, whereas the ionizations governing kcat are not affected. Also, it is seen that the plateau value of kcat is elevated in the presence of chloride, an effect noted by Theorell et al. (32) and subsequently attributed (42) to an increase in rate-limiting dissociation of NADH through displacement of the coenzyme by the chloride anion. Comparable effects were observed in this study for CoLADH, and the results for both metal- enzymes are summarized in Table I.

We have similarly compared the pH and pD dependence of the kinetic parameters governing the oxidation of BzOH. The pD profiles of the kinetic parameters of ZnLADH are illustrated in Fig. 3, and the results are summarized for both metal enzymes in Table I. The pK, values governing kat are uni- formly increased in the presence of D20, and, consequently, the ionization designated by pK2 is not observed for ZnLADH. Correspondingly, for both metal-enzymes, the value of pKz‘ is more influenced by the presence of D20 than is the value of pKl’. In the pH 7-10 region, there is a small but significant deuterium solvent isotope effect (1.8-1.1) on kcat governing the oxidation of BzOH catalyzed by both metalloenzymes. The effect increases at pH values below pKl but decreases at pH values above pK2 according to the isotopically induced shift in each ionization constant. The solvent isotope effect on kCat/KM is of the same magnitude and similarly increases below pKl’ and decreases above pKz’. The parallel nature of the solvent isotope effect for both kcat and kcat/& indicates that the pH-sensitive and the isotope-sensitive processes are the same and determine the parameter value over the range of the pH profile for both metalloenzymes.

Inhibition of Substrate Oxidation by Triflwroethuml-The theory of the influence of hydrogen ions on kinetic parameters


TABLE I Comparison of pK, values of ionizations governing the pH dependence

of the kinetic parameters kCot and kcat& of LADH Ionization constant

Substrate Enzyme kcat t.,lKhi PKI PKZ P K ~ ' PKX'

BzOH ZnLADH" 6.5 (6.9Ib 10.7 8.1 (8.2) 9.6 (10.4) CoLADH" 6.2 (7.1) 9.7 (10.1) 7.7 (8.3) 8.7 (9.3) ZnLADH' 6.7 10.6 7.5 8.9 CoLADH" 6.3 10.0 7.6 7.9 ZnLADHd 6.4 7.6 8.5

2-Propanol ZnLADH" 7.0 >11.5 8.1 >11.5 CoLADH" 6.7 11.0 8.3 10.2 ZnLADH' 7.0 7.3

Ethanol' ZnLADH 6.3 7.4 9.2

Cyclohexanol ZnLADH 6.2 9.0' 7.1' 10.3' 10.5 8.8 10.2

7.0 (8.2)g 9.5 (10.8)#

Measurements were carried out at 21 "C under the conditions of added potassium chloride, as described in the legend to Fig. 2; in general, an error of less than or equal to f 0 . 2 pK. units is estimated for the pK. values determined in this investigation.

pK,, values in parentheses refer to measurements in D20 under conditions of added potassium chloride. e Measurements were carried out in the presence of zwitterionic buffers in the absence of added chloride.

From Ref. 9. The pK. values of ionizations governing b2' are given as pKl' and pK2' since $2' corresponds to the reciprocal of kat/&.

e Calculated by fitting the data of Theorell et al. (32) to theoretical curves. Since these data have not been analyzed previously in this manner, a copy of the calculated pH profile and fit to the data will be provided upon written request.

'From Ref. 11. The second set of entries indicated for this substrate are the ionization constants governing the substrate-activated pathway. In this study, zwitterionic buffers were employed in the absence of added chloride.

From Ref. 39. The pK. values governing kat are not reported. In this study, zwitterionic buffers were employed as in Ref. 11. Values in parentheses refer to measurements in DzO.

shows that kinetically unperturbed ionization constants intrinsic to an enzyme are obtained from the pH dependence of the binding of a competitive inhibitor (36, 37). The pH dependence of the inhibitor constant of TFE for both metalloenzymes has been determined for the oxidation of several alcoholic substrates, a comparison of which is made for the oxidation of BzOH in Fig. 4. The pH dependence of KI reveals ionizations with approximate pK. values of 7.5 and 8.1 for ZnLADH, designated as pKA and pKB, respectively. For CoLADH, the two ionizations are more strongly overlapping. Comparison of the shape of the pH profile of CoLADH to that of ZnLADH shows that the alkaline ionization has been shifted to a lower pK, value, and only an average value of -7.5 can be estimated as the pK. of both ionizing groups. Identical results were obtained for the pH dependence of K, in the oxidation of both 2-propanol and ethanol catalyzed by ZnLADH, confirming that these ionizations are independent of substrate. Such results have not been previously reported in the literature for LADH.

Under "Experimental Procedures," we have described in detail the conditions for investigating the inhibition of BzOH oxidation by TFE, including the order of addition of reagents. Above pH 8, incubation of the enzyme in the presence of NAD+ and TFE, even for the brief period of time required for subsequent substrate addition, led to formation of a weakly fluorescent complex with no detectable change in the absorb- ance of the reaction mixture at 340 nm. Under these conditions, there is an apparent inactivation of the enzyme since the subsequent steady-state velocities upon substrate addition were much lower than when the reaction was initiated by enzyme addition. As illustrated in Fig. 4, incubation of the enzyme with NAD+ and TFE under alkaline conditions leads to obscuring of the ionization pKB = 8.1 that governs the

binding of TFE in the alkaline pH range. Since NAD' is nonfluorescent and no weak fluorescence increase was observed when TFE was added to the enzyme-NAD+ complex at neutral pH, we conclude that the weak fluorescence reflects a protein conformational change within the enzyme-NAD+- TFE complex that is induced only in the alkaline pH range. Fluorine-19 nuclear magnetic resonance studies (43) have revealed that the lifetime of bound TFE in the enzyme-NAD+- TFE complex is 2400 s at pH 2 8.7. Such a long lived bound state is incompatible with behavior as a competitive inhibitor under steady-state conditions, and we, therefore, conclude that this long lived species is responsible for the apparent inactivation of the enzyme at alkaline pH.

pK, values for the ionizing groups controlling TFE inhibition in the oxidation of cyclohexanol and ethanol have been reported by others (11, 13). Our value of pKA = 7.5 agrees directly with their estimates of 7.2 (11) and 7.5 (13). However, Cook and Cleland (11) report a pK, of 10.1 for the ionization governing TFE inhibition of cyclohexanol oxidation in the alkaline range, whereas Shore et al. (13) report no ionization at high pH in the oxidation of ethanol. We note that in both instances kinetic data were collected by absorption measurements rather than by the more sensitive fluorescence method employed here, and no particular mention was made for the order of addition of reagents. We, therefore, conclude that the experimental conditions employed by others (11,13) obscured the pK, of -8.1 through formation of the altered, inactivated complex. Although an ionization controlling kc,,/& in the alkaline range was reported by Cook and Cleland (11) for oxidation of cyclohexanol, their estimates of pKB (10.1) or of pKz' (10.3) do not agree well with the results reported by Taylor (39) for identical substrate and zwitterionic buffer conditions (cf. Table I). Moreover, the earlier steady-state


I k

Om t with KC1 *udfhart KC1

tt lo5 -

IO' -

IO' -

I 02 li

I / /

A I

FIG. 2. Comparison of the influence of pH on the kinetic parameters kat and kaJKM for the oxidation of BzOH catalyzed by ZnLADH in the presence and absence of anions. The pH profiles in the presence of chloride (0) are identical with those for ZnLADH in Fig. 1. The pH profiles in the absence of chloride were obtained by using the following buffers, each at a total concentration of 0.1 M: Pipes at pH 6.1-7.0, Hepes at pH 7.5-8.0, Ches at pH 8.6-9.7, and Caps at pH 10.3-11.0. Other conditions are as indicated in Fig. 1.

results of Theorell et al. (32) yield clear identification of two ionizations governing kcat/KM in the oxidation of ethanol that correspond to pKA and pKB (cf. Table I), as do the results of Dalziel (40).

Influence of Temperature on Ionizations Governing Substrate Oxidation

The temperature dependences of the graphically determined values of pKl and pKl' are shown in Fig. 5. These plots yield estimates of 8.8 and 9.9 kcal/mol for the enthalpies of ionization associated with pKl and pKl', respectively. The circumstance that hydride transfer is rate-limiting in the oxidation of 2-propanol (31) ensures that conformational changes in the protein during coenzyme release should not contribute significantly to these estimates. These results agree with calorimetric estimates (8.8-9.8 kcal/mol) for the enthalpy of ionization of the group with pK, = 7.6 in the enzyme- NAD' complex (44).

In Fig. 6, we have plotted the pH dependence of the enthalpy of activation associated with kcat. We have noted previously (45) that the enthalpy of activation associated with kcat can be separated into a pH-independent term (AH*)li,

associated with ( and the enthalpy of ionization ( A&,) associated with the ionization constant K,. The difference of 8 kcal/mol in Fig. 6 between the limiting values of @ at low pH (16.1 kcal/mol) and at high pH (8.1 kcal/mol) is in good agreement with the value of AHi,, (8.8 kcal/mol) estimated on the basis of the temperature dependence of pKl. Also, the values of AHt in Fig. 6 closely follow a theoretical curve drawn to correspond to the titration of a group with a pK, - 7.0. This is in excellent agreement with the value of pKl obtained

5 6 7 S 9 1 0 1 1 pHup0

FIG. 3. Comparison of the influence of pH and pD on the kinetic parameters kat an&k,. JKM for the oxidation of BzOH catalyzed by ZnLADH. Initial velocity data were collected as described in Figs. 1 and 2 under conditions of 0.1 M potassium chloride with the use of buffers as in Fig. 1. See also "Methods."

from the pH dependence of kc, for 2-propanol, as shown in Table I. The results in Fig. 6 are also in good agreement with the value of A H s of -13 kcal/mol estimated at pH 7.1 on the basis of the temperature dependence of the burst reaction for hydride transfer in the oxidation of ethanol by transient-state kinetic methods (46).

Pocker and Page (47) have reported a value of 9.7 kcal/mol for the enthalpy of activation in ethanol oxidation at pH 7 and have assigned this to the ionization of the hydroxyl proton of metal-bound ethanol. These results determined only at pH 7 cannot be meaningfully compared with the results reported in this study or with those reported by Brooks et al. (46) on the basis of transient-state methods. As shown through Fig. 6, the enthalpy of activation associated with kcat is pH-dependent. Since ethanol oxidation under these conditions is heavily governed by rate-limiting coenzyme release (40, 42, 46), it is doubtful that the result of Pocker and Page obtained under steady-state conditions can be associated with only one chemical process.

In our studies, it has not been feasible to determine the values of the corresponding thermodynamic potentials associated with the ionization constants pK2 and pK2' by steady- state methods since the half-life of the enzyme is <1 min in solutions of pH >12 (48).

DISCUSSION

The pH Dependence of Kinetic Parameters Governing the Oxidation of Alcohols

Assignment of pKl and pK, to the Ternary Enzyme-NAD+- Alcohol Complex-In determining the pH rate profiles of enzyme-catalyzed reactions, the assumption is often made that ionizations governing kinetic parameters behave as unperturbed equilibria. However, this is frequently not the case since the observed ionizations may be shifted from their true


10-41 e

10-6 I T T I I

6 7 8 9 pH

FIG. 4. Comparison of the pH dependence of the inhibitor binding constant of TFE in the oxidation of BzOH catalyzed by ZnLADH and CoLADH. To estimate the pH dependence of the inhibitor binding constant of TFE, only zwitterionic buffers were employed. The inhibitor constants were determined on the basis of initial velocity data collected at the following concentrations of TFE: 1.3 X 4.3 X 8.6 X lo-', and 17.2 X M. The values of Kr are plotted as (0) for ZnLADH and (A) for CoLADH. The ionization constants are indicated by arrows (t for ZnLADH and J for Co- LADH). The values of the ionization constants are p& = 7.5 and pKB = 8.1 for ZnLADH. For CoLADH, only one ionization constant can be determined because of the closely overlapping curves, and we estimate pKA = 7.5 = pKB. Identical results were obtained with 2- propanol and ethanol as substrates, as discussed in the text. The experimental uncertainty in the graphically determined pK, values is approximately k0.2 pK. units. The value of the inhibitor constant at pH 9.5 indicated by an open square (0) was measured under conditions of initiating the reaction by addition of substrate, as discussed in the text. The precise value of the inhibitor constant when measured under these conditions was dependent upon the length of time of incubation of the enzyme with NAD+ and TFE prior to substrate addition, but the general effect of a marked decrease in reactivity towards substrate oxidation was always observed. This result is included here to demonstrate that the ionization for ZnLADH with pK. = 8.1 is obscured under conditions of incubating the enzyme in the presence of NAD+ and TFE prior to substrate addition under conditions of pH > 8. Comparable effects were observed also with other alcoholic substrates.

equilibrium values because of kinetic effects. Our objective in the assignment of the four ionizations governing LADH action has been to first analyze the pH-dependent data for the extent of possible perturbations from equilibrium.

Under conditions of saturating concentrations of NAD', the two-substrate compulsory order, ternary complex mechanism of LADH (38, 49) can be treated as a one-substrate reaction represented in Equation 1.

E-NAD+ + alcohol s E-NAD+-alcohol + E k1 kat

k.1 (1)

+ NADH + aldehyde + H'

For this reaction, the kinetic parameters retain their defini- tions according to theory developed for one-substrate reactions with the understanding that the enzyme-NAD+ complex corresponds to the free enzyme. The pH dependence of the kinetic parameter kc, then depends only on ionizations in the reactive enzyme-substrate complex, ix. the E-NAD'-alcohol complex, under the condition that the reactants and the enzyme-substrate intermediate are in equilibrium (50). Since hydride transfer responsible for ternary complex interconver-

I I

10-61 kcol

FIG. 5. Temperature dependence of the ionization constants pK1 and pK1' governing the kinetic parameters kc., and kcat/ K,, respectively, for the oxidation of 2-propanol catalyzed by ZnLADH. The ionization constants were estimated by fit of the data to theoretical curves calculated for a single ionization. The values and corresponding temperatures for pK1 were 7.22 (14 "C), 7.10 (21 "C), 6.98 (28 "C), and 6.75 (35 "C); forpK1' they were 8.25 (14 "C), 8.00 (21 "C), 7.92 (28 "C), and 7.70 (35 "C). Reaction conditions were otherwise identical with those listed in Fig. 2 for zwitterionic buffers. The enthalpies of ionization estimated on the basis of the least squares slopes are indicated adjacent to each straight line graph.

1s -

AHf

(kcal/mol) 10 x I I I I I I 5 6 7 8 9 1 0

PH

FIG. 6. The pH dependence of the enthalpy of activation associated with kc, for the oxidation of 2-propanol catalyzed by ZnLADH. The data points were visually fitted to a theoretical curve corresponding to a single ionization. The arrow corresponds to a pK. value of 7.0.

sion is the rate-limiting step in the oxidation of 2-propanol (31), application of the theory (50) to Equation 1 then requires that the ionization pK, governing the oxidation of 2-propanol belongs to the enzyme-NAD+-alcohol complex. For the oxidation of BzOH, the values of pK1 agree directly with values of 6.4 in ZnLADH (9) and 6.0 in CoLADH (51) for the ionization governing ternary complex interconversion determined by transient-state methods. Since the values of pK, are similar for both BzOH and 2-propanol despite different rate- limiting steps at higher pH (8, 31, 52), we conclude that the ionization pKl belongs to the ternary enzyme-NAD+-alcohol complex in the oxidation of both BzOH and 2-propanol.

In the alkaline pH range, coenzyme release is slower than hydride transfer in the oxidation of BzOH (9, 52), and the theory for a simple two-step reaction cannot be applied. The ionization pK2 governing the oxidation of BzOH at high pH then could belong to the enzyme-NADH complex, to the enzyme-NADH-aldehyde complex, or to the enzyme-NAD+- alcohol complex. The very large value of the rate constant for aldehyde dissociation compared with that of coenzyme dissociation (8) rules out the enzyme-NADH-aldehyde complex. Furthermore, since no ionization with a pK, < 11.2 governing dissociation of NADH from the enzyme-NADH complex is observed (48), this complex can be also ruled out. We conclude that pK2 with an observed value of 10.7 for ZnLADH reflects an ionization in the enzyme-NAD+-alcohol complex. Under conditions of rate-limiting coenzyme release, the value of pK2


may be modulated by the rate constants for hydride transfer and coenzyme release (37). Accordingly, the intrinsic value of pK2 is expected to be 510.7.'

Assignment of pKl' and pK2' to the Enzyme-NAD+ Com- plex-The pH profile of log kat/KM for both ZnLADH and CoLADH is bell-shaped with an approach to a plateau at high pH, as shown in Figs. 1 and 2. Under conditions of saturating coenzyme as required for Equation 1, the approach to a plateau indicates an ionization in an enzyme-substrate species, whereas the bell-shaped part at lower pH is attributable only to molecular ionization constants of the free enzyme3 in the absence of substrate, i.e. the enzyme-NAD+ complex. The extent of kinetic modulation of pKl' and pKz' governing kcat/ KM can be evaluated by determining their intrinsic values at thermodynamic equilibrium, i.e. through the pH dependence of the binding of the competitive inhibitor TFE under the condition that the ionizations are independent of substrate structure (36, 37). Since this criterion for thermodynamic equilibrium was satisfied in our studies for the binding of TFE, the values of the ionization constants PKA and pKB correspond to unmodulated values intrinsic to the enzyme- NAD+ complex. Comparison of the value of pKA (-7.5) with that of pKl' in Table I for the oxidation of BzOH for identical reaction conditions in the presence of zwitterionic buffers indicates negligible or no kinetic modulation effects. However, the value of pKz' of -8.9 compared with that of pKB = 8.1 shows considerable kinetic modulation. On the basis of relationships derived by Cleland (37) and estimates of rate constants determined by transient-state kinetic methods (9), we calculate pK,' to be shifted to higher values by 20.4 pK, units from its intrinsic value.' The limited data for ethanol oxidation (32) (cf. Table I) suggest that ethanol behaves similarly to the primary alcohol BzOH.

In contrast, slow desorption of 2-propanol in the alkaline pH range would result in a large kinetic displacement of pK2' to values >11, and indeed, pKz' is not observed in the oxidation of 2-propanol (cf, Table I). A similar observation has been made for the secondary alcohol cyclohexanol (11).

A Minimal Reaction Scheme for LADH-Our assignment of two sets of prototropic equilibria to both the enzyme-NAD+ complex and to the enzyme-NAD+-alcohol complex can be employed to define the reaction scheme for the oxidation of alcohols catalyzed by LADH shown as Scheme I. In this scheme, 0 represents the oxidized coenzyme, R the reduced coenzyme, and P the product of alcohol oxidation. The prototropic equilibria governing the initial binding of the coenzyme are drawn in a dashed box to emphasize that our results do not contribute directly to their description. We have indi-

For ionizations observed in the pH profile of kc.,, the influence of kinetic modulation of pKz is estimated from the equation pK2 = pK, + log(1 + k 3 / b ) , where k3 is the rate constant for hydride transfer, b is the rate constant for dissociation of the product NADH, and pKz is the apparent ionization constant as measured through steady-state kinetic studies (37). The equation that applies to ionizations observed in the pH profile of kat/& is ~ K z ' = pK.' + log0 + k3/kZ), where kz is the rate constant for alcohol desorption, k3 is defined above, and pKz' is correspondingly the apparent value of the ionization constant. The values of kz, k3, and b estimated at pH 8.75 on the basis of transient-state kinetic studies (8, 9) have been employed to estimate kinetically induced shifts.

In principle, ionizations governing kcat/& may also belong to the free substrate. However, since the pK, of an alcoholic hydroxyl group is >14 (21), we need concern ourselves only with the enzyme-NAD+ complex. Furthermore, the pH profile of k a t / K ~ reflects ionizations on the free enzyme in the limit of low substrate (alcohol) concentration. Since all of our kinetic data were collected under saturating concentrations of NAD+, the possibility of contributions to kCat/KM from binary enzyme-alcohol complexes can be, therefore, directly disregarded.

SCHEME I

cated also that there are two pathways through which the enzyme-NAD+-alcohol complex can proceed to products because of our analysis of the alkaline region of the pH profile of kCet/KM in Figs. 1 and 2 and because of the pH profile of -log KM, as discussed above.

Differentiation of Metal-induced Shifts in pK, Values from Kinetic Modulation Effects-To assign ionizations of the metal-bound ligands, we verify that changes in pK, values associated with metal ion substitution reflect properties at thermodynamic equilibrium. As discussed above, there is no significant kinetic modulation of the values of pKl and of pKl' since pKA = pKl'. Also, there is no metal effect on this ionization. On the other hand, a kinetic displacement of 20.6 dependent on the rate of hydride transfer is predicted in the value of pK2 for the oxidation of BzOH catalyzed by Zn- LADH.' Correspondingly, the displacement for CoLADH is calculated to be ~ 0 . 5 . ~ Since this kinetic effect would shift the ionization constant from its intrinsic value to higher pK, values and the observed shift in pK2 for CoLADH to lower values is approximately %fold greater than the calculated kinetic shift, we conclude that the decrease in pK2 to a value of -9.7 in the Co'+-enzyme from that of 10.7 in the native enzyme reflects an intrinsic (equilibrium) property induced by metal substitution.

In the enzyme-NAD+ complex, the only metal-influenced ionization is pKz'. The difference in measured values between pKs and pKz' indicates considerable kinetic modulation in the enzyme-NAD+ complex for BzOH oxidation. Also, the modulation is so severe in the oxidation of 2-propanol as to cause an apparent shift to very high pK, values. Nonetheless, even in the oxidation of 2-propanol, the onset of this ionization can be detected for CoLADH with an estimated pK2' - 10.2, whereas the ionization is not detected for ZnLADH (cf. Table I). Most importantly, the intrinsic equilibrium value of the ionization PKB is revealed as a substrate independent property, and the metal-induced shift in the constant is directly demonstrated, as shown in Fig. 4 for TFE inhibition.

Identification of Ionizing Groups Governing Oxidation of Alcohols

The Ionizing Group Responsible for pKl and pKl': Hk-51- Pettersson and co-workers (8-10, 54, 55) have attributed an ionization corresponding to pKl to the metal-bound alcoholic hydroxyl group. We have previously pointed out that the free energy changes associated with the binding of alcohols at low and at high pH are not sufficient to account for a shift in the ionization of an alcohol substrate by 210 pK, units and that linkage relationships require that the ionizing group in the ternary complex responsible for pKl is the same as that in the

~~ ~ ~~

rectly measured for CoLADH, in contrast to ZnLADH (19, 31, 52, 4The rate constant governing hydride transfer has not been di-

53). To provide an estimate of the kinetically induced shift in p&, we refer to the observation that the rate of hydride transfer for ethanol oxidation is decreased from 132 s-l in the native enzyme to 90 s-l in the tetracobalt-substituted enzyme (53).


binary complex observed as pKl‘ (56). In addition, the hy- pothesis of Pettersson and co-workers is now directly contra- dicted by mutagenesis studies. The ionization pKl is abolished in yeast alcohol dehydrogenase through site-specific mutagenesis of His-51 (liver enzyme sequence numbering) to glutamine (57). This ionization is similarly perturbed through mutagenesis of His-51 to glutamine in the human liver Plfll

alcohol dehydrogenase, which has 88% amino acid sequence identity with ZnLADH (58). We conclude that the ionization pKl must have origin in an amino acid residue of the protein.

The salient characteristics of the ionizing group responsible for pKl, as established through these studies, are its insensi- tivity to metal ion substitution and an enthalpy of ionization of -8.8 kcal/mol. For the observed value of pKl = 7.0 and its associated AHi,, of 8.8 kcal/mol, histidine is the only reason- able, metal-insensitive candidate. We assign pKl to His-51, in agreement with others (11, 19). We also attribute pKl’ to His-51 in the enzyme-NAD+ complex. Although this ionization has been attributed previously to the metal-bound water (5, 12-14), we find that pK,‘ and, correspondingly, pKA are insensitive to metal substitution.

It is of interest to note that the presence of the chloride anion induces a detectable change in the value of pKl‘ and pK2’ but not in pKl and pK2, as seen in Fig. 2 and Table I. Since chloride binding to the enzyme-NAD+ complex is competitive with alcohol (41), no perturbation of pKl (or of pKz) should be observed. The shift in the pH profile of k,.,/K, induced by the presence of chloride is, thus, totally consistent with assignment of the ionizations pKl’ (and pK2’) to the enzyme-NAD+ complex. The Ionizing Group Responsible forpK2 andpK2‘: the Metal-

bound Water Molecule-The only ionizations in the pH profiles of the kinetic parameters that are significantly dependent on metal substitution are the ionizations pK2 and pK2’ (or p&). In the enzyme-NAD+ complex, the only metal-linked ionizing group that could be responsible for pK2’ is a metal- bound water molecule? This interpretation is in agreement with a proposal of Cook and Cleland (11) but differs substan- tively from assignments by others whereby only the ionization pKl in the oxidation of BzOH (5, 12) or the ionization with pK. = 7.6 in the enzyme-NAD+ complex have been attributed to metal-bound water (8-10, 14, 54). However, as discussed above, the ionization constants pKl and pKl’ (correspondingly pKA) are insensitive to metal substitution. We also assign pK2 in the catalytically competent enzyme-NAD+-alcohol complex to the metal-bound water molecule, having pointed out earlier that the binding of alcohols is not sufficient to shift the pK, of an alcohol to account for pKl or pK2 (56). Thus, the assignment of pK2 and pKz’ to the metal-bound water molecule and the occurrence of four ionizations controlling the catalytic action of LADH require a substantial reevaluation of the mechanism of LADH in the oxidation of alcohols.

The Mechanistic Role of the Active Site Metal- Water Com- plex in LADH-With assignment of the ionizations pK2 and pK2’ to the metal-bound water molecule, the location of the plateau in the pH profile of kat and the maximum in the pH profile of &/K.M, as illustrated in Figs. 1 and 2, specify that the active site metal ion in the ternary enzyme-NAD+-alcohol complex is coordinated by a neutral water molecule in the physiologically important pH range. With the alcoholic sub-

s Bertini et al. (59) have attributed a pH-dependent decrease in intensity of an NMR band to ionization of the metal-bound imidazole group of His-67 in CoLADH with a pK,, = 9.0. The intensity of this band is shifted and does not disappear with increasing pH in the binary enzyme-NAD+ complex, indicating that the metal-bound imidazole is not responsible for p&’.

strate coordinated to the metal ion in the catalytically active enzyme-NAD+-alcohol complex, as in ternary inhibitor complexes (15, 23-25), the results of these kinetic studies then designate a pentacoordinate alcohol-metal-OH2 complex as the catalytically competent species. We have previously indicated on the basis of electron paramagnetic resonance studies that the binding of coenzyme to CoLADH results in a pentacoordinate complex of the active site metal ion and that the water molecule remains metal-bound in the ternary CoLADH-NAD+-TFE complex (22,60,61).

For a catalytically productive pentacoordinate complex, we suggest that the neutral, metal-bound water molecule serves as a conduit through hydrogen bridging for proton abstraction. In multihydrate metal ion systems, covalent metal-ligand interactions polarize OH bonds and strengthen hydrogen bridging between the metal-bound ligands (62). These interactions can facilitate ionization of the proton from the hydroxyl group of the substrate. Moreover, it has been shown with model metal ion systems that a neutral metal-bound water molecule can have a functional role as a conduit for proton transfer (63). X-ray crystallographic studies (15, 23, 25) of a variety of binary and ternary enzyme-NADH complexes have shown an intricate network of solvent molecules together with the side chains of Ser-48 and His-51 hydrogen- bonded to the ribose OH substituents of the coenzyme. We suggest that the alcohol-metal-OH2 complex in the active site remains tightly hydrogen-bonded to other nearby groups of the proton relay system for kinetically facile proton abstraction and transfer to bulk solvent. Although we have pointed out that there is no significant influence of the metal ion on pKl, it is seen in Table I that the values of pK1 for CoLADH are consistently slightly lower than those of ZnLADH, although the differences are essentially within experimental uncertainty. This observation supports the suggestion that His-51 at the solvent-protein interface “feels” the influence of the open-shell metal ion through a tightly coupled network of hydrogen-bonded residues. This tight coupling is absent in the binary enzyme-NAD+ complex since no parallel metal effect on pKl’ is detected.

It is of interest, furthermore, to point out that the metal- bound water with pK, = 10.7 in the ternary enzyme-NAD+- alcohol complex is ionized under conditions of high pH and that the pH profile of kc,, shows a flattening in this region due to ionization of the alcohol-metal-OH2 complex to an alcohol-metal-OH- species (cf. Figs, 1 and 2). This observation indicates that the ternary complex with a metal-bound hydroxide group must be also catalytically competent, although it is not operant under physiological conditions.

Acknowledgments-We thank Drs. F. J. Kezdy and J. Westley for helpful discussions.

REFERENCES 1. Maret, W., Andersson, I., Dietrich, H., Schneider-Bernlohr, H.,

Einarsson, R. & Zeppezauer, M. (1979) Eur. J. Biochem. 98,

2. Zeppezauer, M. (1983) in Coordination Chemistry of Metalloen- zymes in Hydrolytic and Oxidative Processes (Bertini, I., and Drago, R. S., eds) pp. 99-122, D. Reidel Publishing Corp., Dordrecht, The Netherlands

3. Wolfe, J. K., Weidig, C. F., Halvorson, H. R., Shore, J. D., Parker, D. M. & Holbrook, J. J. (1977) J. Biol. Chern. 252, 433-436

4. Coates, J. H., Hardman, M. J., Shore, J. D. & Gutfreund, H.

5. Schmidt, J., Chen, J., DeTraglia, M., Minkel, D. & McFarland,

6. Welsh, K. M., Creighton, D. J. & Klinman, J. P. (1980) Biochem-

501-512

(1977) FEBS Lett. 84, 25-28

J. T. (1979) J. Am. Chern. SOC. 101,3634-3640

istry 19,2005-2016


7. Morris, R. G., Saliman, G. & Dunn, M. F. (1980) Biochemistry 37. Cleland, W. W. (1977) A&. Enzymol. 45, 273-387

8. Kvassman, J. & Pettersson, G. (1978) Eur. J. Biochem. 87, 417- 2451 427 39. Taylor, K. B. (1983) Biochemistry 22, 1040-1045

9. Kvassman, J. & Pettersson, G. (1980) Eur. J. Biochem. 103, 40. Dalziel, K. (1963) J. Bid. Chem. 238, 2850-2858

19,725-731 38. Wratten, C. C. & Cleland, W. W. (1965) Biochemistry 4, 2442-

565-575 41. Coleman, P. L. & Weiner, H. (1973) Biochemistry 12,1705-1709 10. Pettersson, G. (1987) CRC Crit. Rev. Biochem. 21,349-389 42. Shore, J. D. & Gutfreund, H. (1970) Biochemistry 9,4655-4659 11. Cook, P. F. & Cleland, W. W. (1981) Biochemistry 20,1805-1816 43. Anderson, D. C. & Dahlquist, F. W. (1982) Biochemistry 21, 12. Dworschack, R. T. & Plapp, B. V. (1977) Biochemistry 16,2716-

2725 44. Subramanian, S. & Ross, P. D. (1979) J. Biol. Chem. 254,7827- 13. Shore, J. D., Gutfreund, H., Brooks, R. L., Santiago, D. & San- 7830

tiago, P. (1974) Biochemistry 13,4185-4191 14. Evans, S. A. & Shore, J. D. (1980) J. Biol. Chem. 255, 1509-

45. Makinen, M. W., Kuo, L. C., Dymowski, J. J. & Jaffer, S. (1979)

1514 J. Biol. Chem. 254,356-366

46. Brooks, R. L., Shore, J. D. & Gutfreund, H. (1972) J. Biol. Chem. 15. Branden, C.-I., Jornvall, H., Eklund, H. & Furugren, B. (1975) in 247,2382-2383

The Enzymes (Boyer, P. D., ed) 3rd Ed., Vol. XI, pp. 103-190, 47. Pocker, Y. & Page, J. D. (1990) J. Biol. Chem. 265,22101-22108 Academic, New York 48. Andersson, P., Kvassman, J., Lindstrom, A., Olden, B. & Pet-

16. Klinman, J. P. (1981) CRC Crit. Rev. Biochem. 10,39-78 tersson, G. (1981) Eur. J. Biochem. 113,425-433 17. Dietrich, H., MacGibbon, A. K. H., Dunn, M. F. & Zeppezauer, 49. Cornish-Bowden, A. (1979) Fundamentals of Enzyme Kinetics,

18. Eftink, M. R. (1986) Biochemistry 25, 6620-6624 50. Brocklehurst, K. & Dixon, H. B. F. (1976) Biochem. J. 156,61- 19. Sekhar, V. C. & Plapp, B. V. (1988) Biochemistry 27,5082-5088 70 20. Schneider, G., Eklund, H., Cedergren-Zeppezauer, E. & Zeppe- 51. Sartorius, C. (1987) Die Rolle der Proteinkonformution im Kutu-

zauer, M. (1983) Proc. Nutl. Acud. Sci. U. S. A. 80, 5289-5293 lyzezyklus der Pfurdeleberulkoholdehydrogenuse. PhD thesis, 21. Boyd, R. H. (1969) in Solute-Solvent Interactions (Coetzee, J. F., Universitat des Saarlandes, Saarbriicken, Germany

and Ritchie, C. D., eds.) pp. 97-218, Marcel Dekker, New York 52. Weidig, C. F., Halvorson, H. R. &Shore, J. D. (1977) Biochemistry 22. Makinen, M. W., Maret, W. & Yim, M. B. (1983) Proc. Nutl. 16,2916-2922

Acud. Sci. U. S. A. 80,2584-2588 53. Shore, J. D. & Santiago, D. (1975) J. Biol. Chem. 250, 2008- 23. Cedergren-Zeppezauer, E., Samama, J. P. & Eklund, H. (1982) 2012

Biochemistry 21,4895-4908 54. Kvassman, J. & Pettersson, G. (1979) Eur. J. Biochem. 100, 24. Eklund, H., Samama, J. P., Branden, C. I., Akeson, A. & Jones, 115-123

T. A. (1981) J. Mol. Biol. 146, 561-587 55. Kvassman, J., Larsson, A. & Pettersson, G. (1981) Eur. J. 25. Eklund, H., Plapp, B. V., Samama, J.-P. & Briindhn, C.-I. (1982) Biochem. 114,555-563

J. Biol. Chem. 257, 14349-14358 56. Makinen, M. W. & Maret, W. (1986) in Zinc Enzymes (Bertini, 26. Beaucamp, K., Bergmeyer, H. U. & Beutler, H. 0. (1974) in I., Luchinat, C., Maret, W., Zeppezauer, M., eds.) pp. 465-470,

Methoden der enzymutischen Anulyse (Bergmeyer, H. U., ed.) Birkhauser Verlag, Basel, Switzerland 3rd Ed., p. 580, Verlag Chemie, Weinheim, Germany 57. Plapp, B. V., Ganzhorn, A. J., Gould, R. M., Green, D. W. &

3569-3578

M. (1983) Biochemistry 22,3432-3438 pp. 99-129, Butterworths, London

27. Taniguchi, S., Theorell, H. & Akeson, A. (1967) Acta Chem. Hershey, A. D. (1987) Prog. Clin. Biol. Res. 232,227-236

28. Lowry, 0. H., Passonneau, J. V. & Rock, M. K. (1961) J. Biol. (1991) Biochemistry 30,1062-1068 Chem. 236,2756-2759 59. Bertini, I., Gerber, M., Lanini, G., Luchinat, C., Maret, W.,

29. Dixon, M. (1953) Biochem. J. 55, 161-170 Rawer, S. & Zeppezauer, M. (1984) J. Am. Chem. Soc. 106, 30. Cleland, W. W. (1979) Methods Enzymol. 63, 103-138 1826-1830 31. Brooks, R. L. & Shore, J. D. (1971) Biochemistry 10,3855-3853 60. Makinen, M. W. & Yim, M. B. (1981) Proc. Natl. Acud. Sci. 32. Theorell, H., Nygaard, A. P. & Bonnichsen, R. (1955) Acta Chem. U. S. A. 78, 6221-6225

Scund. 9 , 1148-1165 61. Yim, M. B., Wells, G. B., Kuo, L. C. & Makinen, M. W. (1986) 33. Shore, J. D. & Theorell, H. (1966) Arch. Biochem. Biophys. 117, Frontiers in Bioinorgunic Chemistry (Xavier, A. V., ed.) pp.

34. Dalziel, K. & Dickinson, F. M. (1966) Biochem. J. 100,491-501 62. Zundel, G. (1969) Hydration and Intermolecular Interaction, pp. 35. Dalziel, K. & Dickinson, F. M. (1966) Biochem. J. 100,34-46 76-81, Academic Press, New York 36. Tipton, K. F. & Dixon, H. B. F. (1979) Methods Enzymol. 63, 63. Kluger, R., Wong, M. K. & Dodds, A. K. (1984) J. Am. Chem.

S c u d . 21,1903-1920 58. Ehrig, T., Hurley, T. D., Edenberg, H. J., and Bosron, W. F.

375-380 562-570, VCH Verlagsgesellschaft, Weinheim, Germany

183-234 SOC. 106, 1113-1117

THE OF BIOLOGICAL Vol. 266, No. 5, pp. 20636-20644.1991 Q ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1991 by The American Society for Biochemistry and Molecular Biology, Inc Vol.

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