the journal of chemistry vol. no. 'january 15, pp. k-) u ... · the journal of biological...

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

Vol. 266, No. 2, Issue of 'January 15, pp. 933-941.1991 Printed in U. S. A.

Activation and Inhibition of Bovine Carbonic Anhydrase I11 by Dianions"

(Received for publication, May 29, 1990)

Roger S. Rowlettz, Nicholas J. Gargiulo 111, Frank A. Santoli, Jill M. Jackson, and Anita H. Corbett From the Department of Chemistry, Colgate University, Hamilton, New York 13346

We have found that many dianionic species, at millimolar concentrations, significantly activate or inhibit the bovine carbonic anhydrase 111-catalyzed hydration of COz. Dianionic species such as HP0:- and SO",, with pKa values near 7, are activators, whereas weakly basic species such as SO:- act as inhibitors. Both activation and inhibition are partial hyperbolic in nature and do not appear to compete with monoanionic linear inhibitors like N:.

Our kinetic data are consistent with a formal mechanism of action for carbonic anhydrase I11 that is directly analogous to that of carbonic anhydrase 11, in which Lys-64 of carbonic anhydrase I11 can act as an intramolecular H' transfer group during COz hydration. Our data suggest that dianionic inhibitors depress the rate of H+ transfer during turnover by stabilizing the protonated form of Lys-64. We postulate that dianionic activators enhance the rate of a rate-limiting H' transfer step in the mechanism, probably by acting directly as H' acceptors.

Carbonic anhydrase (carbonate hydrolyase, EC 4.2.1.1) is an enzyme that catalyzes the reversible reaction:

CO, + H,O + HCO, + H' (1)

At least three genetically distinct cytosolic CA' isozymes are known to exist in vertebrates, which, although sharing homologous structures, have greatly different catalytic proper- ties (Silverman and Lindskog, 1988). All three CA isoenzymes contain a catalytically essential Zn2+ ion, which is coordinated to 3 His residues; a fourth coordination site on the Zn" ion is presumed to be occupied by water. X-ray crystallography reveals that the Zn2+ ion occupaes the bottom of a roughly conical cavity approximately 15-A deep in isoenzymes I (Kan- nan et al., 1975) and I1 (Eriksson et al., 1988), and a similar, but more constricted cavity in isoenzyme I11 (Eriksson, 1988).

Erythrocyte CA I1 is perhaps the best understood, as well as the most efficient of the carbonic anhydrases, with a maximal turnover of approximately 1 X lo6 s-' (Khalifah, 1971). A large body of kinetic data has led to the general acceptance of a zinc hydroxide mechanism for CA 11, in which the conversion of CO, into HCO: by Zn2+-bound hydroxide is separated in time from the release of H' (Steiner et al., 1975).

* This work was supported by Grant 19508-B3 (to R. S. R.) from Petroleum Research Fund, administered by the American Chemical Society, and by National Science Foundation Grant DMB-8617161. 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.

$ To whom correspondence should be addressed. ' The abbreviations used are: CA, carbonic anhydrase; MES, 2-(N-

morpho1ino)ethanesulfonic acid MOPS, 3-(N-morpholino)pro- panesulfonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine.

A simplified version of such a mechanism for the CO, hydration reaction is shown in Equations 2 and 3:

EZn-OH + CO, $ EZn-HCO: (2) + H 2 0 == GZn-OH, + HCO;

EZn-OH2 + EZn-OH + H' (3)

In CA 11, the pK, of the Zn2+-bound water molecule is approximately 7.0, limiting the H+ transfer rate constant to H 2 0 to about 1 X lo3 s-'. Thus, for this isoenzyme it is clear that the release of H' in Equation 3 must be assisted in some way by either internal or external buffers, in order to account for the observed maximal turnover of 1 X lo6 s-'. It has been shown that buffer molecules are the eventual acceptor of the hydrogen ions generated by Equation 3 (Jonsson et al., 1976), and that the rate of intermolecular H+ transfer approaches diffusion control at high pH (Rowlett and Silverman, 1981). Yet, CA I1 displays a solvent deuterium isotope effect in kcat of approximately 3.8, even at high pH where the intermolecular, buffer-mediated H' transfer is occurring at diffusion- controlled rates, implying there is some other rate-determining H' transfer step in the mechanism (Steiner et al., 1975). It has thus been postulated that His-64, which is positioned near the opening of the active site cavity of CA 11, acts as an intermediary in transporting H' ions out of the active site during turnover. This intramolecular H' transfer reaction is apparently the rate-limiting step in overall catalysis (Silver- man and Lindskog, 1988). Direct evidence for the importance of His-64 in the catalytic mechanism comes from site-specific mutagenesis studies: human CA I1 in which His-64 is replaced by Ala has a kcat value for C02 hydration of only 1.6 X IO4 s-', approximately 5% of that of the native enzyme (Tu et al., 1989).

CA 111, found in skeletal muscle tissue, is structurally homologous with CA I1 (Eriksson, 1988). The most obvious structural difference between bovine CA I11 and CA I1 is the substitution of Lys-64 in the former for His-64 in the latter. In addition, CA I11 contains a larger proportion of basic residues in and around the active site than CA 11. For example, CA I11 has Arg residues at positions 67 and 91, whereas the corresponding residues in CA I1 are Asn and Ile. Amino acid positions 64 and 67 lie near the opening of the active site cavity in both CA I1 and CA 111. Perhaps because of the concentration of positive charge in the active site, the pK, of the Zn"-bound H 2 0 is observed to be less that 6.0 in this isoenzyme (Ren et al., 1988b). Bovine CA I11 also contains 5 Cys residues, compared to one in human CA 11, but the role of these residues in catalysis, if any, is not known.

These structural differences have a pronounced effect on the catalytic ability of CA I11 as compared to CA 11. The reported maximal kc,, value for feline CA I11 is estimated to be only 1 X lo4 s-' (Kararli and Silverman, 1985), 100 times

933

934 Activation and Inhibition of Carbonic Anhydrase 111

slower than for CA 11. In addition, the maximal rate of C02/ HCO, exchange (Equation 2) measured by 13C NMR at chemical equilibrium is reported to be only 1.2 x lo4 s" for CA I11 (Ren et al., 1988a), compared to 3 X lo6 s" for human CA I1 (Simonsson et al., 1979). However, despite these differences, CA I11 is thought to share a mechanism (Equations 2 and 3) similar to the other mammalian CA isoenzymes. Specifically, CA I11 is thought to be rate-limited by a H+ transfer step based on several lines of evidence: Kararli and Silverman (1984, 1985) observe that the maximal rate of CO,/HCO; exchange at pH 7.0 is much larger than the observed kcat value of 2000 s-', and that kc,, has a significant solvent deuterium isotope effect; Ren et al. (1988b) observe that during CO, hydration by Co'+-substituted bovine CA 111, a species accumulates at steady state whose visible spectrum is consistent with enzyme-bound Co2+-H20.

Despite the relative abundance of kinetic and structural data concerning CA 111, no detailed catalytic mechanism has been worked out that is consistent with all of the extant data. Indeed, several recent investigators have noticed unusual kinetic behavior in CA I11 catalysis. Ren et al. (1988a) reported non-Michaelis-Menten kinetics in the HCO, dehydration reaction, and anomalous progress curves in the C02 hydration reaction. Silverman and Tu (1986) report biphasic kinetics in the CO,/HCO; exchange reaction at chemical equilibrium in "0 isotope exchange experiments. Finally, Shelton and Cheg- widden (1988) reported the activation of the HCO, dehydration reaction of human and chicken CA I11 by HPOe- and so:-.

We have examined the interaction of bovine CA I11 with a variety of dianionic species, including HP0:- and SO:-. We have found that many dianions are hyperbolic activators or inhibitors of the enzyme, and act in the millimolar concentration range. In particular, we have found that SO:- is a significant inhibitor of bovine CA 111. In the absence of SO:-, we have determined the maximal kcat of the enzyme is near 3 X IO4 s-', significantly larger than previously reported. The kinetic data we have collected is consistent with, and can be reasonably interpreted using, a mechanism of action directly analogous to that of CA I1 (Rowlett, 1984; Lindskog, 1984), in which Lys-64 acts as an intramolecular H+ transfer group, albeit an inefficient one. We posit that dianionic inhibitors or activators exert their effect by altering the rate of H+ transfer in the catalytic mechanism.

EXPERIMENTAL PROCEDURES

Materials-MES, MOPS, Bicine, chlorophenol red, m-cresol purple, and p-nitrophenol were obtained from Sigma. Distilled, doubly deionized water with a resistivity of 12 megohms cm-' or greater was used throughout to minimize contamination by adventitious metal ions. All other reagents were of ACS grade or better, were obtained from commercial sources, and used without further purification.

Enzyme-Bovine CA 111 was obtained from flank steak and purified using a modification of the method of Tu et al. (1986). All operations described below were carried out at 4 "C. One kilogram of cubed flank steak was homogenized in small batches in a blender with a total of 1-2 liters of 10 mM Tris/H,S04 buffer, pH 8.0, containing 1 mM mercaptoethanol. The total blending time for each batch was about 5 min. The resulting product was centrifuged at 4000 X g for 30 min, and the supernatant pooled and filtered through cheesecloth. To this solution was added 600 g/liter of (NH&S04, and the resulting suspension centrifuged a t 11,000 X g for 30 min. The precipitate was resuspended in deionized water containing 1 mM mercaptoethanol, and dialyzed exhaustively against the same. This crude enzyme solution was filtered through Whatman No. 1 filter paper, and concentrated to 40 ml with 20 mM Tris/HnSOd, 0.1 M Na2S04, 1 mM mercaptoethanol, pH 8.0. The clear protein solution was passed through a 6 X 50-cm gel exclusion column (Ultrogel AcA 44, LKB) using the concentration buffer. The active fractions, as determined by the method of Rickli et al. (19461, were pooled and

concentrated to 25 ml in 10 mM Tris/HzS04, 1 mM mercaptoethanol, pH 8.5. This solution was applied to 2.5 X 50-cm ion exchange column (DEAE-Sephacel, Pharmacia LKB Biotechnology Inc.) and eluted with the concentration buffer. The active fractions comprising the major protein peak were pooled and concentrated in 1 mM mercaptoethanol.

The enzyme concentration was estimated from the absorbance at 280 nm, based on a molar absorptivity of 6.2 X lo4 M" cm" (Engberg et al., 1985). Sodium dodecyl sulfate-gel electrophoresis of the purified preparation showed one major protein band of molecular weight 30,000. Typically, 500-600 mg of purified CA 111 can be obtained from 1 kg of flank steak. The protein can be stored in 1 mM mercaptoethanol solution at 4 "C for many months without significant loss of activity.

Kinetic Methods-Saturated solutions of CO, were prepared by bubbling CO, gas into water in a vessel maintained at 25 & 0.1 "C, and dilutions were prepared in the absence of air by coupling two gas-tight syringes as described by Khalifah (1971). COZ concentrations were calculated based on a 33.8 mM (H20) or 38.1 mM (D,O) saturated solution a t 25 "C (Pocker and Bjorkquist, 1977).

The progress of CO, hydration or HCO: dehydration was measured by the changing pH-indicator method (Khalifah, 1971) using a Hi- Tech SF-4 stopped flow spectrophotometer equipped with a Zenith 2-159 microcomputer and Metrabyte DAS-16 rapid data acquisition system. The portion of the flow circuit containing the reactants (about 500 pl) and the observation cell were immersed in a thermostated fluid bath maintained at 25 k 0.1 "C.

Buffer-indicator pairs used, their approximate pK. values (25 "C), and the wavelengths monitored in kinetic runs were MES (pK. = 6.1) with chlorophenol red (pK, = 6.3, 574 nm), MOPS (pK, = 7.2) with p-nitrophenol (pK, = 7.1, 400 nm), and Bicine (pK, = 8.3) with m- cresol purple (pK. = 8.3, 578 nm). In a typical stopped flow experiment, one drive syringe contained a COZ solution, and the other drive syringe contained buffer, enzyme, and indicator dye. These two solutions were mixed in the stopped flow in COz:buffer ratios varying from 1:l to 5:1, depending on the maximum final COZ concentration desired.

Analysis of Kinetic Data-The reaction velocity, u, was obtained from a logarithmic plot of the progress curve according to Equation 4 (Lindskog and Thorslund, 1968), where Q is a buffer-dependent factor relating changes in absorbance to changes in [H+], M is the total absorbance change for the reaction, At is the absorbance at time t, and A,, is the absorbance a t equilibrium:

u = QM (d In(A, - A,,)/&) (4)

The term d In(At - A,,)/& was obtained from a linear least squares analysis of a plot of h (A , - AeJ uersus time. This method of obtaining reaction velocities, rather than the conventional initial rate method, was used because, in some instances, the enzyme-catalyzed reaction required a small but significant time period to reach steady state. All reaction velocities were corrected for the uncatalyzed reaction, which typically comprised less than 20% of the overall rate. Kinetic param- eters kcat and K,,, were determined by nonlinear least squares regres- sion analysis similar to that described by Cleland (1967).

NMR Experiments-All "P NMR line broadening experiments were performed with a Bruker AC-250 NMR spectrometer equipped with a 10-mm broadband probe, a t a frequency of 101 MHz. All experiments were carried out at 21 "C, and in the presence of 10% D20 to maintain frequency lock. No 'H decoupling was employed. In a typical experiment, 1024 scans were averaged into 16 K data points. Linewidths of 31P peaks were determined by a nonlinear least squares analysis program supplied with the NMR spectrometer, using samples with the highest practical enzyme concentrations. Typical standard errors in determined linewidths were k0.2 Hz.

Measurements of enzyme catalyzed C02/HCOi exchange rates were carried out as described previously (Simonsson et al., 1979, 1982), with two modifications. First, the intrinsic linewidth of the HCO; resonance, used to correct all measured linewidths, was estimated from experimental mixtures in which the enzyme was com- pletely inactivated with 10 mM azide, rather than acetazolamide. Second, correction of observed linewidths for hound substrate was not necessary under our reaction conditions (pH 7.0, 199 mM [COZ] + [HCO,], 149 p M enzyme).

Computer Simulations-Computer simulations of the proposed bovine CA I11 mechanism were performed according to Hurst (19671, using Microsoft QuickBasic 4.5 on a Zenith 2-159 microcomputer

Activation and Inhibition of Carbonic Anhydrase III 935

equipped with an 8087 numeric coprocessor. Sensitivity indices were calculated according to Ray (1983).

RESULTS

Sulfate ion was found to be a partial hyperbolic inhibitor of CO, hydration catalyzed by bovine CA I11 (Fig. 1). The data are satisfactorily modeled by Equation 5 , which describes partial hyperbolic inhibition:

u ~ / u ; = (1 + P[SO:-]/KJ/(l + [SO:-]/K,) (5)

Here, uo/u, is the velocity of the reaction catalyzed by the uninhibited enzyme divided by the velocity at a given concentration of SO$-, p is the maximal -fold inhibition, and K; is the apparent dissociation constant for SO:-. When maximally inhibited, the enzyme is reduced to 58% of its original activity, and the apparent dissociation constant for Na2S04 is quite small, about 1.1 mM. With MgS04 as the SO:- source, the maximal inhibition was somewhat less, to 72% of the original activity, and with an apparent dissociation constant of only 0.7 mM. We have no adequate explanation for this observation, but note that Simonsson and Lindskog (1982) observed similar behavior in the weak SO:- inhibition of human carbonic anhydrase I1 at low pH.

Sulfate concentrations up to 16 mM were observed to have no significant effect on the C02/HCO: exchange rate, measured by 13C NMR at pH 7.0,199 mM [CO,] + [HCO:].

We found sodium phosphate to be a hyperbolic activator of the CO, hydration reaction catalyzed by bovine CA 111 (Fig. 2). The data are adequately modeled by Equation 6, where u,/ U o is the velocity of the reaction catalyzed by the phosphate- activated enzyme divided by the velocity of the unactivated enzyme, p is the maximal -fold activation, [PI is total phosphate concentration, and K, is the apparent dissociation constant for total phosphate.

UdUn = (1 + P[PI/KJ/(l + [Pl/KJ (6)

The maximal activation is about 6.5-fold, with an apparent dissociation constant of 1.0 mM. The amount of activation is substantially greater than, and the apparent dissociation constant for total phosphate considerably less than, suggested by the data of by Shelton and Chegwidden (1988) for the CA I11 catalyzed HCO; dehydration reaction.

v o / v i

t 1.04 c

0 2 4 6 E 10 12 14 16

[ s u l f a t e l (rnM1

FIG. 1. Inhibition of bovine CA 111-catalyzed COZ hydration by NazS04 (A) and MgS04 (0). Reaction conditions were: pH 7.0; 50 mM MOPS, 50 p M p-nitrophenol, 4.23 mM CO,, 2.4 p M enzyme; 25 "C. Curves drawn through the data were obtained from a least squares fit to Equation 5. For Na2S04, B = 1.7, K; = 1.1 mM; for MgS04, (3 = 1.4, K,. = 0.70 mM.

[phosphate] h M )

FIG. 2. Activation of bovine CA 111-catalyzed COz hydration by sodium phosphate. Reaction conditions were: pH 7.0; 50 mM MOPS, 50 p M p-nitrophenol, 4.23 mM CO,, 2.4 p M enzyme; 25 " c . Curve drawn through the data was obtained from a least squares fit to Equation 6, obtaining 0 = 6.5, Kp, = 1.0 mM.

v o / v i

E" 0

0 0

2"

04 c 0 2 4 6 E 10 12 14 16

[sulfatel (mM1

FIG. 3. Inhibition of bovine CA 111-catalyzed COz hydration by sodium sulfate in the presence of sodium azide, 0 mM (A), 200 HM (a), 400 pM (0). Reaction conditions were: pH 7.0; 50 mM MOPS, 50 p M p-nitrophenol, 4.23 mM coz, 2.4 p M enzyme; 25 "c. Curves through the data are based on Equation 7, where = 1.6, Ki. = 0.84 mM, and Ki. = 100 pM.

Phosphate concentrations up to 25 mM were found to have little effect on the C02/HCO; exchange rate, measured by 13C NMR at pH 7.0,199 mM [CO,] + [HCO;].

To learn more about the binding site of SO:-, we measured bovine CA 111-catalyzed CO, hydration rates in mixtures of SO:- and N:, the latter ion a known inhibitor of CA I11 (Kararli and Silverman, 1985). We find that the inhibitory effect of SO:- is not diminished by inhibitory concentrations of N; (Fig. 3). These two inhibitors appear to act independently, SO:- as a partial hyperbolic inhibitor, and NT as a classical linear inhibitor. We have modeled the data using Equation 7, which describes independently acting partial hyperbolic (SO:-) and linear (N;) inhibitors:

un/u, (1 + [NyI/Ka)(l + P[SOFI/Kd/(1 + [SO&-[/KiJ (7)

In Equation 7, uo/u; is as defined in Equation 5, (3 is the maximal -fold inhibition by SOf, and Ki, and Ki, are the apparent dissociation constants of N: and SO$-, respectively.

In contrast, phosphate and SO:- do not appear to be acting independently on the enzyme, according to measurements of

936 Activation and Inhibition

bovine CA 111-catalyzed COz hydration rates in the presence of mixtures of phosphate and SO:- (Fig. 4). Apparently, high concentrations of SO:- can overcome phosphate activation, and inhibit the enzyme to a maximal extent similar to that of SO:- alone. These data are not satisfactorily modeled by Equation 7 but are favorably modeled by a mutually exclusive partial hyperbolic activator/inhibitor pair, described by Equa- tion 8:

uo/u, = (1 + P[so:-]/Ki + [P]/Kp/(l + [so:", + O1[P]/Kp) (8)

In Equation 8, u$ui is as defined in Equation 5 , [PI is the total phosphate concentration, p is the maximal -fold inhibition by SO:-, a is the maximal -fold activation by phosphate, and Ki and K, are the apparent dissociation constants of SO:- and total phosphate, respectively.

Additional evidence for the mutual exclusivity of phosphate and sulfate interaction with bovine CA 111 come from 31P NMR measurements. A small but significant line broadening of the 31P NMR signal from aqueous sodium phosphate is observed in the presence of bovine CA 111. We assume that phosphate is in fast exchange with the enzyme, since we observe only one 31P signal for phosphate in the presence of enzyme. With increasing concentrations of added Na2S04, the

linewidth narrows to the limit of that of free phosphate, consistent with the kinetically measured apparent equilibrium dissociation constants of total phosphate and SOZ- (Fig. 5 ) . The data of Fig. 5 were modeled using Equation 9, which describes the expected linewidth of an NMR signal of a nucleus in fast exchange between two environments:

wobs = fbwb + ffwf (9)

In Equation 9, Webs is the observed linewidth, f b and f f are the fractions of enzyme-bound and free phosphate, respectively, and w b and wi are the linewidths of enzyme-bound and free phosphate, respectively. That the data can be successfully modeled using the kinetically determined dissociation constants of SO:- and phosphate suggests we are not monitoring the displacement of nonspecifically bound phosphate from the enzyme.

We found that kinetic data for the SO:- inhibition of the bovine CA I11 catalyzed CO, hydration reaction generated uncompetitive inhibition patterns throughout the pH range 6.5-9.5, although the maximal extent of inhibition was found

* 1.5

0.0 2 4 6 8 10 12 14 16

[sulfatel (mM)

FIG. 4. Inhibition of bovine CA 111-catalyzed CO, hydration by sodium sulfate in the presence of sodium phosphate, 0 mM (A), 200 FM (0), 500 PM (0). Reaction conditions were: pH 7.0; 50 mM MOPS, 50 p M p-nitrophenol, 4.23 mM co,, 2.4 pM enzyme; 25 "c. Curves through the data are based on Equation 8, where P = 1.7, 01 = 6.5, K, = 1.0 mM, and Kp. = 1.0 mM.

of Carbonic Anhydrase 111

5 .44 c o 5 10 15 20 25 30 35

[sulfate] ImM)

FIG. 5 . Sulfate dependence of the 31P NMR linewidth of sodium phosphate in the presence of bovine CA 111. Experimen- tal conditions were: pH 7.0; 50 mM MOPS, 1 mM total phosphate, 150 p~ bovine CA 111; 21 "C. Curve drawn through the data is based on Equation 9, where w b = 25 Hz, wf = 5.5 Hz, and fb, ff were calculated based on dissociation constants of 1.0 mM for both phosphate and SO:-.

l/ LC021 ( l / m M l

FIG. 6. Lineweaver-Burk plot of inhibition of the bovine CA 111-catalyzed COz hydration by sodium sulfate; 0 mM (O), 50 mM (A). Reaction conditions were: pH 7.0; 100 mM MOPS, 50 pM p-nitrophenol, 4.3 p M enzyme; 25 "c. For [so:-] = 0, kcat = 2600 s-', K,,, = 14 mM; for [so:-] = 50 mM, kcat = 1500 s-', K,,,. = 8 mM.

to decrease markedly at higher pH values. A typical plot is shown in Fig. 6. As seen in Fig. 6, steady state rates of CO, hydration followed Michaelis-Menten kinetics, as did HCOT dehydration (data not shown). For HCO; dehydration, the K,,, of HCO: was too high to allow precise determination of kcat, but we observe a kCat/Km value of 1.4 X lo4 "' s" at pH 7.0.

In addition to SO:- and phosphate, many other dianionic species were found to be partial hyperbolic inhibitors or activators of bovine CA 111. Table I is a summary of these results, with the pKb of the dianionic species, the maximal -fold inhibition or activation, p, and the apparent dissociation constant, Kdiss, listed for each ion.

From Table I, it appears that inhibition or activation of bovine CA I11 is not specifically accomplished by phosphate or SO:- but can be achieved by a wide variety of dianionic species. Whether a particular anion is an activator or inhibitor seems to correlate with the pKb of the dianionic species: most weak bases are activating, but the weakest bases are inhibi-


TABLE I Some partial hyperbolic inhibitors and activators of bovine CA I l l The following conditions were used pH 7.0; 50 mM MOPS, 50 pM

p-nitrophenol, 1.2 p M enzyme; 25 "c. Substance PKb" Action B &is.

HP0:- Imidazole so2- Maleate Malonate Oxalate so:- SeOf

6.9 6.9 7.1 8.0 8.3

10.0 12.1 12.1

Activator Activator Activator Activator Activator Inhibitor Inhibitor Inhibitor

mM

6.5 1.0 3.4 94 1.6 0.11 2.3 0.75 2.7 1.1 3.1 51 1.6 1.0 1.6 2.0

Obtained from Weast (1988).

r

0

I 0 A I

log k c a t 4 . 0 I 6.0 6.5 7.0 7.5 8.0 8 . 5 9 .0 9.5 10.0

pH

FIG. 7. pH profile of COz hydration catalyzed by bovine CA I11 in the presence of 0 mM sodium phosphate, 0 mM NazSOl (El), 60 mM NazSOr (A), 50 mM sodium phosphate (0). Reaction conditions were 100 mM MES, 50 /IM chlorophenol red, pH 6.5, 100 mM MOPS, 50 p M p-nitrophenol, pH 7.0-7.5, or 100 mM Bicine, 25 p M m-cresol purple, pH 8.0-9.5,25 'c, and 0.5-1.8 p M enzyme. Except for 50 mM sodium phosphate, curves drawn through data were obtained by least squares fit to Equation 10. For 0 mM sodium phosphate, 0 mM NazS04, kin = (1.8 & 0.4) X lo3 s", kmax = (3.2 & 0.3) X lo4 s-', pK. = 7.97 & 0.10; for 50 p M NazS04, k,i, = (1.3 f 0.2) X lo3 s-', kmaX = (2.1 f 0.4) X lo' SKI, pK, = 8.81 f 0.18; for 50 mM sodium phosphate, kcar = (3.9 & 0.2) X lo4 s-'.

tory. At least one neutral species is capable of activation, namely imidazole, although a larger concentration is required to activate compared to dianions. In contrast, none of the large molecular weight buffers (MES, MOPS, Bicine) used in this study showed any significant activation or inhibition over the 10-100 mM concentration range. We also did not observe any activation by cacodylate, (CH&AsO;, in the 1-50 mM concentration range.

The pH-rate profile of bovine CA I11 is greatly affected by the presence of SO:- or phosphate. The pH profile of kcat for COZ hydration catalyzed by bovine CA I11 (Fig. 7), in the absence of activating or inhibitory anions, appears to follow a pH titration curve with a transition from a small, but significant, minimum value of kcat at low pH, to a maximum value of kc,, at high pH (Equation 10).

kobs = kmin + (kmax - k m i n ) / ( l + [H+I/Ka) (10)

In Equation 10 kobs is the observed value of kcat at any given pH, kmi, is the minimum, limiting value of kcat at low pH, k,,, is the maximum limiting value of kc,, at high pH, and K, is the apparent acid dissociation constant controlling the transition. The uninhibited, unactivated enzyme appears to be

controlled by an apparent pKa close to 8.0, whereas the maximally SO:- inhibited enzyme follows a similar pH titration curve with a controlling pKa displaced about 0.8 pH unit upward. However, when bovine CA I11 is maximally activated by (saturating) phosphate, the pH profile of kcat becomes pH- independent, with an observed kcat value close to the maximal kcat value for the enzyme in the absence of phosphate or so:-.

We have also measured the solvent deuterium isotope effect on kc, and k J K , for COz hydration, in the absence of SO:- and phosphate, at pH 7.0 and pH 9.0 (Table 11). We found that the magnitude of the isotope effect on kat diminished at the higher pH, while the isotope effect on kcat/Km was similar at the two pH values. While our measured isotope effect on kcat/& at pH 7.0 is similar to that previously reported (Kararli and Silverman, 1985; Ren et al., 1988), our measured value of the isotope effect on kcat at the same pH value is much larger.

DISCUSSION

We contend that all of the data reported here is accounted for by a proposed kinetic mechanism of bovine CA 111, shown in Scheme 1, in which Lys-64 plays an important role as a H' transfer group. Scheme 1 is essentially identical to the cur- rently accepted mechanism of CA I1 (Steiner et al., 1975; Lindskog, 1984; Rowlett, 1984), in which His-64 plays the role of H+ transfer group. We further hypothesize that activation and inhibition of bovine CA I11 by dianions are a consequence of the alteration of the rate of H' transfer during catalysis, Our data favor, but probably do not conclusively demonstrate, a distinct binding site for inhibitory and activatory dianions. We describe below in detail the proposed mechanism, and its

TABLE I1 Solvent deuterium isotope effects of steady state kinetic constants for

COZ hydration catalyzed by bovine CA III Reaction conditions used were: 100 mM MOPS, 50 p~ p-nitro-

phenol, pH 7.0, or 100 mM Bicine, 25 p M m-cresol purple, pH 9.0; 25 "C; pH is the uncorrected pH meter reading. The superscript H indicates that the measurement was carried out in H20, and D indicates that the measurement was carried out in D20.

pH (kdH/(k.dD ( L J K m ) H / ( k d K m ) D 7.0 9.0

6.0 f 0.9 1.5 ? 0.2 3.5 f 0.5 1.6 & 0.2

kloH*

SCHEME 1. Proposed mechanism of action for bovine CA 111. Here Zn is the active site Zn2+ ion and Lys is the Lys-64 side chain. Proposed rate constants are given in Table 111.


ability to accommodate the experimental data we have collected. The latter has been done primarily through computer simulations of Scheme 1 a t steady state using a digital computer. We also reflect on additional ramifications of our proposed mechanism that may possibly be further experimentally tested.

The Proposed Mechanism-The mechanism of Scheme 1 involves the following basic elements and is qualitatively described below. First, we assume that the active form of the enzyme for COz hydration is that in which the Znz+-bound water molecule is ionized to give a hydroxide ion. This Zn2+- bound OH- can attack CO, to yield a Znz+-bound HCOg complex (steps kl or k5). The HCO, can be displaced by water, yielding a ZnZ+-H20 complex (steps k3 or ks). The Zn2+-Hz0 complex may then lose H' by one of the two pathways; 1) the H+ can be released directly to H,O (step &), or 2) H+ may be transferred to Lys-64, (step kll), and then donated to buffer molecules in solution (step k13). Clearly, the second of these H+ transfer pathways is not significant except a t higher pH values, where a significant fraction of Lys-64 is deprotonated, and buffer molecules are sufficiently basic to act as efficient H+ acceptors. Thus, the involvement of Lys-64 in the mechanism of CA I11 offers an explanation for the source of the pH dependence of kcat. We hasten to add that large molecular weight buffer molecules apparently cannot directly accept H+ from the Zn2+-Hz0 complex, because we do not see activation by buffers such as MOPS, MES, and Bicine. This conclusion is consistent with the fact that the active site cavity of bovine CA I11 is considerably more constricted than in CA I1 (Eriks- son, 1988).

Although there is only a small body of kinetic data for CA I11 (nearly all of which is somewhat compromised by the inclusion of large amounts of SO:-, which we have shown here to be inhibitory), the extant data does provide useful limits on the magnitude of rate constants in a mechanism such as Scheme 1. Our approach was to use the existing kinetic data, and that reported here, to assign reasonable values for some of the rate constants in Table 111, and to assume chemically and physically reasonable values for the remainder of the rate constants, adjusting them as needed to quantitatively simu- late steady state kinetic data. We used a similar approach to accurately model the kinetics of CA I1 (Rowlett, 1984). Al- though this method does not guarantee a unique solution for the rate constants of a kinetic scheme, we note that Lindskog

TABLE 111 Proposed rate constants for Scheme 1

The rate constants of this table are consistent with a pK, of 6.0 for Znz+-bound HzO and 9.0 for Lys-64.

Rate constant Value Units

k , 3 x lo6 k, 3 x lo4 S-'

k3 3 x lo4 S-I

k4 1 x lo6 ks 1 x lo6 126 3 x 104 S-l

k7 3 X 104 S-I

ka 1 x lo6 k, 2 X 103 S-I

klo 2 X 109 4 x lo5 S-l

kl2 4 x 10, S"

k r " 1 x 109

"I s-l

"I s-I

"1 s-l

"I s-I

"I s-l

krnax0 13 1 X 109 "I s-l

"I s-I

a Maximum value of this rate constant; the actual value at any pH depends on the K, values of Lys-64 (KLJ and the buffer (Khuff), according to the equation kohs = kmax/(l + Kbuff/KLys) (see Rowlett, 1984).

(1984), working independently on a computer model of the mechanism of CA 11, arrived a t values of rate constants very similar to ours (Rowlett, 1984).

Some kinetic limitations on the mechanism of action of CA I11 are well agreed upon, and we have adopted these in Scheme 1. Two lines of evidence suggest that the pK, of Zn2+-bound water in CA I11 is 6.0 or less. First is the pH independence of the visible spectrum of Co'+-substituted bovine CA I11 (Ren et al., 1988b). Second is the uncompetitive mode of inhibition of CA I11 by N, throughout the pH range 6.0-9.5 (Kararli and Silverman, 1985); uncompetitive inhibition patterns are observed for Ng inhibition of CA I1 at alkaline pH, when the Zn2+-bound water is largely deprotonated (Pocker and Deits, 1982). Both of these results suggest that the Zn2+-bound water is largely deprotonated in the pH range 6.0-9.5.

The enzyme appears to be limited in rate, at least partially, by a H' transfer step based on 1) rapid-scan stopped flow spectrophotometry (Tu and Silverman, 1986; Ren et al., 1988b), which indicates that a metal-bound water complex accumulates a t steady state in the CO, hydration reaction, and 2) a significant solvent deuterium isotope effect on kc,, noted by us and others (Kararli and Silverman, 1985; Ren et al., 1988a). These two observations specifically imply that the regeneration of Zn2+-bound hydroxide is at least partially rate-determining in CO, hydration. This conclusion is further amplified by measurements of the maximal rate constant of C02/HC0g exchange, (kCcaJexch. Ren et al. (1988a) report a

value of 1.2 X lo4 s-I, using a 13C NMR technique, while Silverman and Tu (1986), using an isotope exchange method, estimate (keaJexch to be near of 2 x lo4 s-'. Our own 13C NMR results set a lower limit of approximately 7 X lo3 s" for

Accordingly, we have assigned to the Zn2+-bound water a pK, of 6.0 in Scheme 1. While others have suggested a much lower pK, for the metal-bound water molecule (Ren et al., 1988b. Kararli and Silverman, 1985), we found that a pK, of 6.0 was more satisfactory than lower values in modeling our pH profiles of kcat (Fig. 7) and kJK, (data not shown). However, Scheme 1 is still in qualitative, if not quantitative, agreement with the observed data with a slightly lower pK, for the metal-bound water molecule. We also note that the ratio kZk3/(k2 + k3) in Scheme 1 provides an upper limit for the value of (kcaJerch. The rate constants of Table I11 yield a k2k3/(kz + k3) ratio of 1.5 X lo4 s-', consistent with the experimentally observed values of (kCcaJerch. We have assigned the step labeled k, as the rate-limiting step of the CO, hydration reaction, with a value of 2 x IO3 s-'. We note that the size of this rate constant is consistent with H+ release from a Zn2+-H20 complex with a pK, of 6.0, assuming a diffusion controlled association of H+ and the Zn2+-OH complex with a rate constant of 2 X lo9 s-I. This latter rate constant agrees with the estimated rate constant for the diffusion- controlled association of buffer molecules and human CA I1 (Rowlett and Silverman, 1982). Another constraint on the rate constants of Table I11 also applies, namely that the ratio kll/klz = KZJKL,,, where KZn and KLrs are the acid dissociation constants for Zn2+-Hz0 and Lys-64, respectively. The other rate constants of Table I11 were adjusted so that computer simulations of COz hydration produced kcat and K, values in general agreement with experiment. We did find, during simulations of Scheme 1, that it was necessary to set k1 > kg, in order to account for the slight pH dependence of kCat/Km, (data not shown). Also, we found that simulations of Scheme 1 using the rate constants of Table I11 gave the best fit to the observed data (Fig. 7) when the pK, of Lys-64 was set to approximately 9.0. That is, the apparent pK, observed in the


pH profile of kc,, does not correspond to the actual pK, of the titrating group. We noticed, by carrying out many computer simulations of Scheme 1 while varying the rate constants of Table 111, that the apparent controlling pK, was always lower than the true pK. of the titrating group. Thus, the kinetically observed pK, must represent a lower limit for the true pK, of the titrating group. We note that a pK,, of 9.0 is certainly consistent with the expected pK, of a Lys side chain. Using the rate and equilibrium constants of Table 111, we find that Scheme 1 predicts a pH profile of kcat that varies from a low pH value of 2 X lo3 s" to a high pH value of 3 X lo4 s-' with an apparent pK, value of 8.0, in close agreement with Fig. 7.

A qualitative assessment of the source of the pH dependence of kcat in the mechanism of Scheme 1 is as follows. At low pH, the enzyme is rate-limited in C02 hydration by the release of H' from Zn2+-H20 to bulk water. This is a consequence of Lys-64 being largely protonated, and unable to accept H" for a more rapid, buffer-mediated transfer to bulk solution. With increasing pH, as the fractional deprotonation of Lys-64 becomes larger, and solution buffers are increasingly basic, the competence of the Zn2+-Hz0 - Lys-64 + solution buffer H' transfer pathway is enhanced. At the high pH plateau, this pathway becomes so facile that another step in the mechanism, perhaps HCO: release, becomes rate-limiting for k,,,.

Phosphate Activation-An appealing notion to explain the activation of bovine CA I11 is the participation of phosphate as a H' transfer agent, bypassing the Lys-64 H' shuttle. We are encouraged in making this interpretation based on "0 isotope exchange experiments which show for bovine CA I11 the rate of loss of water bearing substrate oxygen is considerably enhanced in the presence of phosphate? This process thought to be at least partially rate-limited by H+ transfer (Silverman and Tu, 1986). We note that the monohydrogen phosphate ion, HPOZ-, has a pKa near 7.0, and is thus ideally suited as a H' transfer group, being both a good H' acceptor and donor in aqueous solution. We envision two possible ways in which HP0:- can accelerate H' transfer: 1) HPOi- can accept H' from protonated Lys-64 or 2) HPOf can accept H' directly from the Zn'+-bound water molecule.

Case 1 is difficult to justify, based on the observation that not all buffers are capable of activating the enzyme. If HP0:- can accelerate H+ transfer from Lys-64 to bulk solution, then surely all buffers can. The side chain of Lys-64 is well exposed to solution and should be able to interact with buffers without steric hindrance, yet we do not observe activation by the buffers MES, MOPS, and Bicine, nor do we observe any activation by cacodylate. Nevertheless, we carried out computer simulations of Scheme 1 in which the rate of H' transfer from protonated Lys-64 was enhanced by HPO&- in a concentration-dependent way. These simulations predicted K, values for COS that were at least an order of magnitude higher than the experimentally observed K, values of approximately 20-30 mM in the presence of 50 mM phosphate.

On the other hand, simulations of case 2, in which HP0;- can directly accept H' from Zn2'-bound water, faithfully mimics the observed magnitude pH independence of kc,, (Fig. 7 ) , and furthermore, predicts the experimentally observed slight increase in K, for CO, with increasing pH. For the purposes of computer simulation, we replaced step lzg in Scheme 1 with the rate constants (lzg + klSIHPOe-]) and step klo[H'] with the rate constants (klo[H+] + k16[H2P0;]), where kls and k16 are effective rate constants for the binding and H+ transfer steps of phosphate-mediated H+ transfer. We found

C . K. Tu and D. N. Silverman, personal communication.

that any values for k,, and kI6 greater than 5 X lo6 "' s-' produced computer-simulated pH profiles of kc,, that were essentially pH-independent with a value near 3 X lo4 s-' over the pH range 7.0-9.5.

The qualitative assessment of the pH independence of CO, hydration in the presence of phosphate is as follows: the HP0:"mediated H' transfer is so efficient compared to the normally available pathways, that the enzyme is rate-limited over the entire pH range by some step in the CO,/HCO: interconversion sequence.3 This modified version of Scheme 1 does not distinguish between the cases where 1) HP0:- accelerates H' transfer in an encounter-controlled reaction, or 2) HP0:- has a distinct binding site on the enzyme, near the active site, and is essentially acting as a prosthetic H+ transfer group. However, we tend to favor the second explanation, in which there is a nonspecific dianion binding site, for reasons that will be detailed later.

Sulfate Inhibition-Unlike HPOi-, SO:- is an extremely weak base that is incapable of assisting in H' transfer. Indeed, the uncompetitive inhibition pattern observed for SO:- in the CO, hydration reaction (Fig. 6), tends to suggest that SO:- selectively binds to the Zn2+-H20 form of enzyme that accumulates during steady state turnover of COz. This is the interpretation of similar kinetic patterns seen for monoanion inhibition of CA I1 (Rowlett, 1984; Lindskog, 1984; Tibell et al., 1984). However, that explanation seems to be inadequate here. First, monoanions are classical linear inhibitors of CA 11; SO:- is a partial hyperbolic inhibitor of bovine CA 111. Second, we have kinetically demonstrated that SO:- and N;, the latter a typical linear inhibitor of bovine CA 111, do not appear to compete for the same form of enzyme during catalysis; otherwise inhibition of CA I11 by mixtures of SO:- and N; would not be multiplicative (Fig. 3). Third, we note that SO:- is not observed to inhibit the CO,/HCO; exchange reaction, as measured by isotope exchange' or 13C NMR techniques. Thus, we rule out the binding of SO:- to the metal ion, and must look for another explanation. We favor an explanation in which SO:- stabilizes the protonated form of Lys-64, shifting its pK, upward. The pH profile of the SO:- inhibited enzyme is consistent with computer simulations of Scheme 1 where the pK, of Lys-64 is shifted upward 0.8 unit to 9.8. Thus, at any given pH value, the enzyme has a lower kc,, in the presence of SO:- than in its absence, and we find that both kcat and K,,, are lowered in the presence of SO$- by about the same factor: i.e. the simulated inhibition patterns are uncompetitive. Our explanation accounts for the observation that the maximal inhibition by SO:- diminishes at high and low pH extremes. We propose that SO:- binds to bovine CA I11 at a locus where it is in close proximity to Lys- 64, electrostatically stabilizing the positively charged, protonated form of the side chain. Thus, it appears that SO:- may simply increase the pH required to make the Lys-64-mediated H' transfer pathway competent.

Generality of Dianion Activation and Inhibition-Based on the mechanism of Scheme 1, and our assessment of phosphate activation and sulfate inhibition, it is possible to make the following predictions. First, dianionic species whose pKb = 7.0 (good H' acceptors and donors), should be capable of activating bovine CA 111. Second, dianionic species whose pKb >> 7.0 (poor H+ acceptors) should not be activators, but

There is no pH dependence of phosphate-activated carbonic anhydrase 111-catalyzed CO, hydration because the pH profile of Fig. 7 was collected in the presence of suturuting phosphate. Thus even at low pH, there is sufficient HP0:- to maximally activate the enzyme, and no dependence of kcet on the pK, of HzPOa is expected or observed.


inhibitors, due to their perturbation of the pKa of Lys-64. Thus in Table I we note that HPOi-, SO$, maleate, and malonate are activators of CA 111, all with pKb values within 1.5 pH units of 7.0, whereas the weakly basic oxalate, SO:-, and Se0:- are all inhibitors.

For human carbonic anhydrase 11, it has been shown that the rate of proton transfer from enzyme to buffer species (as measured by k c a t / E where K", is the apparent K, of the buffer species) is essentially independent of the buffer structure, and dependent on the pKa difference between the buffer and enzyme in a manner following a Bronsted relationship, with a maximal k c a t / z near the diffusion-controlled limit (Rowlett and Silverman, 1982). While the number of activating species reported here for CA I11 is too small to be treated meaningfully in this manner, we do note the following facts. First, in our small sample of activators the value of k,,/Kdi,, for CA I11 activators is structure-dependent: HP0:- and imidazole both have a pK, of 7.1 for their conjugate acids, but the relevant kCat/Kdi,, values are 2.6 X lo7 and 1.4 X lo' M" s-', respectively. This latter value compares well to the reported value of 1.2 X lo5 "' s-' for the rate of H' transfer between human CA I11 and imidazole measured by an isotope exchange method (Tu et al., 1990). MOPS, with a conjugate acid pK, also near 7.1, is not an activator at all. Second, while the magnitude of kCat/Kdi,, follows the sequence SO$ (5.8 X lo7 M" s-') > HP0:- > maleate (1.3 X lo7 M-' s-') > malonate (9.8 X lo6 M" s-'), in fair agreement with the sequence of pK, values of the conjugate acids, the values of kceat/Kdiss are much smaller than that expected of rate constants for near- diffusion-controlled processes. For the activators so far stud- ied, both the magnitude of the H+ transfer rate constants, and the structural specificity for activation, argues against buffer species acting as encounter-controlled H+ transfer agents in a way analogous to that for human CA 11.

The Case for a Dianion Binding Site-Several observations indirectly support the idea of a specific dianion binding site on the enzyme. First, we note that the concentration of dianion required to activate or inhibit the enzyme is quite small, typically in the millimolar range, as reflected by the apparent dissociation constants of Table I. While it is possible to explain dianion activation solely in terms of an encounter- controlled H+ transfer reaction, it is exceedingly difficult to account for SO:- inhibition except in the way that we propose. For example, we can rule out significant binding of SO:- to the Zn2+-Hz0 form of the enzyme during COz hydration, because SO:- does not appear to inhibit the CO2/HCO; exchange reaction, nor does SO:- compete with N,, an anion which has been amply demonstrated to inhibit in this mode (Tibell et al., 1984; Kararli and Silverman, 1986). We can also rule out as a cause of inhibition the bulk ionic strength effects of the investigated anions, because significant inhibition is observed under conditions where the ionic strength increase due to the added anion salt are only a few percent. For example, in a typical sulfate inhibition experiment, in which 100 mM MOPS buffer, pH 7.0, is present (ionic strength =0.05), the enzyme-catalyzed COz hydration rate is reduced by 25% in the presence of 1 mM Na2S04, a salt concentration which raises the ionic strength of the medium by only 0.003, or 6%.

Second, we note that at least one other low molecular weight buffer with a pK, near 7.0, namely imidazole, is also an activator of bovine CA 111, but only at high concentration. Compared to typical dianionic activators, a concentration of imidazole nearly 100 times greater is required to have a similar effect. Neutral imidazole and HPOq- have identical basicities

and are similar in molecular volume; the only distinguishing characteristic is that HP0:- is charged and imidazole is not. We hypothesize that HP0:- binds to a positively charged locus near the active site of the enzyme, where it can effi- ciently accept H', directly or through intervening water bridges, from Zn2+-bound water. A likely site is near Lys-64 and Arg-67, both of which lie at the opening of the active site cavity. A binding site for HPOP and other activating dianions explains the low concentrations of these substances required to activate the enzyme. By contrast, neutral imidazole cannot be bound or directed to the active site, and a much larger concentration is required to have a similar activatory effect.

Third, we note that SO:- is capable of displacing phosphate in both kinetic experiments (Fig. 4) and NMR experiments (Fig. 5) with the same apparent Ki value. So, although the NMR experiment cannot distinguish between kinetically significant and kinetically insignificant phosphate binding, the fact that in both experiments the same apparent Kt for SO:- is obtained suggests that we are monitoring the same phosphate displacement process. In both experiments, we observe significant displacement of phosphate by millimolar concentrations of SO:-. Under these conditions, the ionic strength change from added sulfate is only a small fraction of the total ionic strength contributed by buffer species. Thus it appears that we are observing a true displacement process, and not just ionic strength effects.

Thus, although the evidence is indirect, we favor the idea of a distinct binding locus for dianions, near the active site, separate and distinct from the Zn2+ ion, the binding site for monoanions like N:. An attractive candidate for this binding site is near Lys-64 and Arg-67. Dianions binding here would be poised for H+ transfer (for basic anions like HPOi-), or appropriately positioned to perturb the pKa of Lys-64 (as for

One consequence of this idea is that the extent of dianion binding should be sensitive to the ionic strength or concentrations of competing counterions, and this may be the case. We note that much larger concentrations of sulfate or phosphate are required to inhibit or activate the HCO, dehydration reaction, respectively, compared to the COz hydration

This effect is most notable at large concentrations of HCO,, and is less noticeable as the HCO; concentration decreases. In the HCO, dehydration reaction, of course, there is an unavoidably high concentration of NaHC03; in the COz hydration reaction, the substrate has no ionic strength con- tribution.

Physiological Significance-There is probably no specific physiological significance for dianion activation or inhibition of CA 111. As we have shown, many dianionic bases with a pKb near 7 will activate CA 111, including a variety of phys- iologically relevant organic phosphates.' At typical concentrations of organic phosphates ( 4 0 mM) for mammalian muscle tissue, and at physiological pH (-7.4) and ionic strength (~0 .15) (Hawk et al., 1954), where the apparent dissociation constant for phosphate activation is near 50 mM,4 we estimate the apparent activation of CA I11 to be approximately 3-fold. CA I11 will not be appreciably inhibited by SO:- in muscle, but it will be significantly inhibited by C1-, which is present at approximately 12 mM (Hawk et al., 1954). Using the reported Ki for C1-, 6 mM (Sanyal, 1984), we estimate that CA I11 is inhibited 3-fold by this anion in muscle (i.e. 33% active). Thus, overall, we expect CA I11 to have about the same activity in muscle tissue as in in vitro experiments without added anionic inhibitors.

so:-).

R. S. Rowlett, personal communication. D. N. Silverman, personal communication.

Activation and Inhibition of Carbonic Anhydrase I I l 941

TABLE IV Sensitivity indices of kcat for the CO, hydration reaction according to

Scheme I Sensitivity index"

pH 6.0 pH 7.0 pH 8.0 pH 9.0

kl 0.001 0.000 0.001 0.001 ka 0.001 0.000 0.051 0.435 k.5 0.001 0.000 0.001 0.001 k7 0.074 0.163 0.506 0.434 ks 0.775 0.281 0.023 0.001 kl1 0.151 0.541 0.412 0.128 k n 0.003 0.005 0.007 0.001

Rate constant

a Indices do not add to exactly one due to roundoff.

Implications of the Proposed Mechanism-There are some consequences of Scheme 1, and of our hypotheses of activation and inhibition, that are experimentally verifiable. One such consequence is the pH dependence of the solvent deuterium isotope effect. The magnitude of the solvent deuterium isotope effect on kc,, is expected to be determined by the extent with which H' transfer steps are rate-limiting in overall turnover. We have used the treatment of Ray (1983), in which intrinsic sensitivity factors for each step in the mechanism of Scheme 1 can be accurately calculated. The sensitivity factors represent the fraction of the intrinsic isotope effect in that mech- anistic step that will be expressed in a measurement of the solvent deuterium isotope effect on kc,,. The sensitivity factors for all the forward (or reverse) steps of a kinetic mechanism add up to exactly one; the larger the sensitivity factor, the more "rate-limiting'' that step is for kcat. Sensitivity indices for the CO, hydration reaction according to Scheme 1 are shown in Table IV.

It is clear from Table IV that those steps in the mechanism in which there is a H' transfer (4, kll, and kI3) are largely ( ~ 9 3 % ) rate-limiting at pH 6.0, whereas these steps are not significantly ( ~ 1 3 % ) rate-limiting at pH 9.0. Thus, one ex- pects a significant decrease in the observed solvent deuterium isotope effect on kcat on going from pH 6.0 to 9.0.6 We observe (Table 11) a decrease of isotope effect on kc,, of almost 2-fold on going from pH 7.0 to 9.0, in qualitative accordance with the model.

Table IV also points out a major theme of Scheme 1: the pH dependence of the mechanism represents a shift of rate determining step from H' transfer at low pH, to CO,/HCO; interconversion steps at high pH. The pH at which that transition occurs depends on the pK, of Lys-64. Alternatively, the pH dependence can be abolished in the presence of an activatory dianion.

We fully expect that the proposed mechanism of CA I11 in Scheme 1 will be tested using site-specifically mutagenized CA 111. We predict from Scheme 1, for example, that a CA I11 in which Lys-64 is replaced with Ala-64: 1) will show a pH- independent k,, of =2 X lo3 s-'; 2) will not be inhibited by SO:-; and 3) may or may not be activated by low concentrations of HPO:-, depending on the importance of Lys-64 to our proposed dianion binding site. We also predict similar

fi Without detailed knowledge of the intrinsic isotope effect on each of the 13 forward steps in the proposed mechanism, it is of course impossible to predict the exact expected isotope effect on kcat. How- ever, assuming that only steps k, k , , , and kI3 have significant isotope effects, and that kH/kD = 6, then the predicted isotope effect on kcat will decrease from approximately 6 to 2 on going from pH 7.0 to 9.0.

results for equine CA 111, which is quite homologous with bovine CA 111, the only apparently important distinction being the replacement of Lys-64 with Arg-64. Our proposed anion binding site locus might be profitably probed using CA I11 mutants in which Lys-64, Arg-67, and Arg-91, or combina- tions of these are altered. If any of these mutations alter our proposed anion binding site, then the concentrations of activators or inhibitors required to exert an effect will be in- creased. We are engaged in some of these studies now.

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