THE Joumia~ OF BIOLWK!AL CHEMISSTRY Vol. 242. No. 22, Issue of November 25, pp. 6212-5219, 1967

Printed in U.S.A.

Mechanism of Action of Carbonic Anhydrase

SUBSTRATE, SULFOKAMIDE, AND ANION BINDING*

JOSEPH E. COLEMAN

(Received for publication, May 4, 1967)

From the Department of Biochemistq, Yale University, New Haven, Connecticut 06510

SUMMARY

Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II), and Hg(I1) form metallocarbonic anhydrases containing these metal ions at the same binding site. Of this series, only the Zn(I1) and Co(I1) enzymes catalyze the hydration of CO2 and the hy- drolysis of p-nitrophenyl acetate. Likewise, only Zn(I1) and Co(I1) induce the metal ion-dependent binding of 3H-aceta- zolamide at the active center. Acetate, azide, cyanate, sulfide, and cyanide compete with 3H-acetazolamide for the binding site at the active center. Binding of 1 eq of sulfide and cyanide to the Zn(II) and Co(I1) enzymes at pH values below the pK, values of H2S and HCN is accompanied by the release of H+ ions. Over the pH range in which the in- hibitors are in the form CN- and HP, binding is accom- panied by the appearance of -OH ions. The experimental data fit the theoretical curves constructed by assuming a single additional H+ dissociation associated with the protein, coupled with the metal ion, and described by a pK, of 8.1. The latter coincides with the inflection point of the pH-rate profile for catalysis by human carbonic anhydrase B. Apo- and the inactive metallocarbonic anhydrases do not show the alterations in hydrogen ion equilibria accompanying the addi- tion of CN-. Accompanying spectral data as a function of pH show that -OH, HC03-, and CN- compete for the same binding site on the Co(I1) enzyme. A mechanism based on a mixed enzyme-zinc-hydroxide as the active form of the enzyme is proposed for the hydration of CO2 and the hydroly- sis of p-nitrophenyl acetate.

Carbonic anhydrase from mammalian erythrocytes was the first zinc metalloenzyme to be discovered (1,Z). The properties of the highly purified enzyme with a molecular weight of about 30,000 have been investigated in a number of laboratories (3-13) through studies which have shown the existence of several isozymes of the enzyme, the dependence of catalytic activity of

* This work was snpported by Grant AM-09070-03 from the National Institutes of Health, United States Public Health Serv- ice.

all isozymes on the presence of the metal ion, the inhibition by a variety of sulfonamides (14, 15) and metal-binding anions, and the existence of at least three reactions catalyzed by the en- zyme; the hydration of COZ, the hydration of certain aldehydes (16), and the hydrolysis of several esters (13, 17-20). The present paper reports equilibrium studies on the mechanisms of binding of metal ions, sulfonamides, and anions at the active site of carbonic anhydrase. Data on the relationship of these mechanisms to the catalytic activity of the protein are discussed.

EXPERIMENTAL PROCEDURE

Reagents-All chemicals were reagent grade. Buffer solutions, HCl, NaOH, substrates, and indicators were prepared metal-free as previously described (13, 21, 22). Spectrographically pure metals or their salts (Johnson Mattey Company, Ltd.) were used to prepare Mn(II), Co(II), Ni(II), Cu(II), Cd(II), and Hg (II) carbonic anhydrases. Acetazolamide (2-acetylamino-1,3,4- thiadiazole-5-sulfonamide, Diamox) was kindly supplied by Dr. E. H. Dearborn of the Lederle Laboratories.

Enzymes--Human carbonic anhydrases, isozymes B and C, were prepared as previously reported (8, 12). Macoca mulatto carbonic anhydrases B and C were isolated in crystalline form as described in Duff and Coleman (13). Bovine carbonic anhydmse B was purchased as a lyophilized preparation (Seravac Labora- tories) prepared by the method of Lindskog (3) using zone electrophoresis for final purification. The material was sub- jected to further column chromatography on DEAE-Sephdex and the peak fractions were used for the present experiments. Additional samples of bovine enzyme were prepared from bovine red cells by methods similar to those described for the human and simian enzymes (3, 12, 13). APO- and metallocarbonic anhy- drases were prepared as previously reported (12, 13).

Enzymatic Activities-Hydration of CO* was measured by the method of Rickli et al. (8) and Wilbur and Anderson (23), with bromothymol blue as indicator. With this method, human and monkey isozymes B have activities of about 10,000 U, while the two C isozymes have activities of about 30,000 U (Wilbur- Anderson units). Activities of apo- and metallocarbonic anhydrases assayed by this method were as previously reported (12, 13).

Esterase activity was measured with the use of p-nitrophenyl acetate as the substrate (17) following the absorbance change at

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400 rnp or 348 mp, the latter being an isosbestic point for p- nitrophenol and p-nitrophenolate (24). Unless specified, the reaction cuvette contained 1 X lop3 M p-nitrophenyl acetate in 0.025 M Tris-0.02 M acetate, 5% acetonitrile. Enzyme concen- trations in the assay varied from 3 to 5 x lo-” M. The pH was varied from 5 to 10.5. The buffer combination used depended upon the species of metal ion present., and for the zinc enzyme 0.025 M Tris-0.02 M Na acetate was routinely used. Tris, up to 0.25 M, was noninhibitory, while higher concentrations of acetate inhibited. The pa-rate profiles run with the Zn(I1) enzyme in Tris alone were identical with those in Tris-acetate. The Co(I1) enzyme reaction mixture contained Tris alone in view of the potent inhibition of the Co(I1) enzyme by acetate (see “Results and Discussion”).

Protein concentrations were determined from measurements of the optical densities at 280 rnp with molar absorptivities of 4.90 x lo4 M-I cm-’ for human B and 5.34 X lo4 M-’ cm-l for human C (25); 4.88 X lo4 M-~ cm-l for monkey B and 5.35 X lo4 M-~

cm-l for monkey C (13) ; and 5.60 X lo4 M-’ cm-l for bovine B

(3). Visible absorption spectra of the various metallocarbonic

anhydrases were obtained on a Cary model 15 recording spectro- photometer or a Perkin-Elmer model 350 recording spectro- photometer. Path lengths were 1 cm.

Binding of e6Zn and 3H-Acetazolamide to Carbonic Anhydrase- The Sephadex procedure used to measure binding of radioactive metal ions to proteins was that outlined in Coleman and Vallee (26). Binding experiments with tritiated acetazolamide were performed by the equilibrium dialysis technique of Coleman and Vallee (27) with the use of 3H-acetazolamide (New England Nuclear) labeled in the acetyl group. The material migrated as a single peak on a radiochromatogram. The counting procedure was modified by the use of a scintillation mixture of naphthalene and dioxane (28) which permits the direct introduction of aqueous samples without prior drying. Counting was done at 4” in a Tri-Carb liquid scintillation spectrometer (Packard model 3002).

Complexometric Titration-The complexometric or difference titration method used for detecting the release of H+ or -OH from carbonic anhydrase on interaction with anionic inhibitors is similar to the pH-stat method developed by Coleman and Vallee (29) for the measurement of proton release accompanying the binding of metal ions to proteins. The instruments used were Radiometer titrators (type TTTlc) equipped with titragraph recorders (type SBR2c). In the present method, both inhibitors and enzyme were contained in unbuffered solutions initially titrated to the same pH. When working with HCN and H2S at the lower pH values, great care must be taken to avoid change in the pH through loss of HCN or HzS gas before the reaction is started. In a closed electrode vessel, 5 ml of a 0.01 M solution of cyanide or sulfide were adjusted to the required pH immediately before the reaction. A O.l-ml aliquot was then transferred to the electrode vessel containing the protein at the same pH and the H+ or -OH released titrated with dilute NaOH or HCl. Ap- proximately 1 pmole of protein (30 mg) was used for the determi- nation of each point, and the acid and base concentrations were so adjusted that the release of one H+ or -OH ion per protein molecule (1 pmole of H+ or -OH) required titration with about 0.1 ml of tit)rant (0.01 N). Volumes of the reaction mixture varied from 3 to 5 ml, protein concentrations from 2 to 4 X lop4 M. In order to take full advantage of the sensitivity of the

inst,rument and improve the precision of the method, the drive for the micrometer syringe was fed through a series of reduction gears such that full scale deflection on the titragraph recorder corresponded to 43 ~1 instead of 500 ~1. Precision of the method was ho.02 pmole of H+ in the low pH range, f 0.05 pmole of H+ at pH 9 or above. Metallocarbonic anhydrases other than Zn(I1) were prepared by adding equimolar metal ions contained in 0.002 M citrate, pH 7.0, to the apoenzyme. The preparations were then dialyzed against metal-free water to remove the last traces of buffer. The Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(H), and Hg(I1) enzymes were analyzed by atomic absorp- tion spectroscopy with a Jarrell-Ash spectrometer and were found to contain between 0.9 and 1.1 g atoms of metal per mole of protein.

RESULTS AND DISCUSSION

e5Zn Binding to Human Carbonic Anhydrase B: Interference by Mn(Zl), Ni(Zl), Co(ll), Cu(ll), Cd(ZZ), and Ilg(ZZ)-Of the first transition and IIB metal ions, only Zn(I1) and Co(I1) have been shown to restore CO2 hydration or esterase activity to apocarbonic anhydrase. The other divalent metal ions of this series restore no or minimal activity (12, 13,30). Exchange data using e6Zn to displace enzyme-bound cobalt at pH 5.0 have indicated that Zn(I1) and Co(I1) occupy the same binding site (5). X-ray structure data show that the Hg(I1) binding site is close to if not identical with the Zn(I1) site (11). No data have been presented, however, to indicate that the whole series of metal ions occupy the same binding site. Fig. 1 shows that the firm binding of the first transition and IIB metal ions to carbonic anhydrase is mutually exclusive. If equimolar f15Zn is added to the apoenzyme, and the mixture passed over a G-25 Sephadex column, the label binds tightly to the protein and emerges in the excluded volume (Fig. 1A). I f one of the first transition metal ions beginning with Mn(I1) or a IIB metal ion is added prior to 65Zn, the label does not bind to the protein and is held up by the column (Fig. 1B). Thus, these ions all appear to occupy the same site as the Zn(I1) ion.

In order to test the complementary conclusion that the pro- t,ein-containing fractions in the excluded volume contained 1 g atom of firmly bound stable isotope, suitable aliquots from the mixtures used for the experiments in Fig. 1B were passed over the Sephadex column and the metal content of the fractions was determined by atomic absorption spectroscopy. Three representative examples, nickel, cadmium, and mercury, are shown in Fig. 1C. The size of the aliquots was varied to insure concentrations of each metal ion well within the sensitivity range of the method. Metal-containing fractions correspond to the protein-containing fractions in each case, and the metal content is proportional to protein content. Total metal recovery per mole of protein, calculated from the total absorbance at 280 rnp and the metal concentration per ml, was 0.91, 0.97, and 0.98 g at.om per mole for the mixtures containing Ni(II), Cd(H), and Hg(II), respectively. Similar findings pertain to the other metal ions used.

Thus, the different visible absorption spectra (12, 30, 31), the differential binding of sulfonamides (5, 20) and the differential activities (20, 30) reported for this set of metallocarbonic anhy- drases must reflect the particular coordination chemistry charac- teristic of the interaction of each of these ions with the same site. In addition to the restriction of significant catalysis of the hydra- tion of CO2 and the hydrolysis of p-nitrophenylacetate to Zn(I1)

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Mechanism of Action of Carbonic Anhydrase Vol. 242, No. 22

8 C.

FRACTION NO. (2ml)

FIG. 1. Prevention of 05Zn(II) binding to human apocarbonic anhydrase I3 by Mn(II), Co(IIj, Ni(II), Cu(IIj, Zn(II), Cd(II), and Hg(IIj. One-milliliter samples of 1 X 1Om6 M apocarbonic anhydrase plus 1 X 10e5 M eSZn(II) (A), and 1 X 10--6~ apocarbonic anhydrase plus 1 X 1OW M Me(I1) (Me(I1) = Mn(II), Co(II), Ni(II), Cu(II), Cd(II), or Hg(l1)) plus 1 X 10e5 M BSZn(IIj (B) were passed over a G-2.5 Sephadex (coarse bead) column (1 X 30 cmj at a flow rate of l-ml per min. Absorbance at 280 rnp was monitored with a Gilford scanning device (model 2000) and the optical density trace is indicated by the vertical lines. 65Zn(II) ( l j was determined on each 2 ml of eflluent and is expressed as counts per min per 2-ml fraction. The properties of the coarse bead form of G-25 Sephadex are such that ionic 66Zn diffuses rapidly and appears as a long low peak, 2 to 500 cpm above back- ground. Complete recovery requires 200 fractions. 6, aliq,uots corresponding to 7.1 mg of the Hg(I1) enzyme, 4.5 mg of the Nl(I1) enzyme, and l.G mg of the Cd(II) enzyme were taken from the solu- t,ions used in B and passed over the Sephadex column. Optical density at 280 mp (A, El, 0) and metal content (A, W, l ), in micrograms per ml, were determined on each 2-ml fraction; (a, A) Ni(I1) enzyme, (0, l ) Cd(I1) enzyme, and (0, n ) Hg(IIj enzyme. The buffer system for all experiments was 0.025 M Tris, pH 8.0, 23”. The inner left ordinate refers to the optical density of the Hg(I1) enzyme fractions.

and Co(I1) carbonic anhydrase (20, 30), these are the only two

ions which induce the metal ion-dependent binding of the sul- fonamide inhibitor, 3H-acetaeolamide, to carbonic anhydrase

(20). Anion and Hydrogen Ian Eflects on Sulfonamide Binding-

Monodentate metal-binding anions have been assumed to inhibit carbonic anhydrase by adding to an open coordination site of the metal ion or replacing a ligand already present. In support of this postulate, binding of all anions tested affects the energies and

symmetry of the d orbitals of the metal ion as indicated by the shift in wave length and optical activity of the d-d absorption

bands of the Co(I1) enzyme (5, 12, 31). These anionic inhibitors interfere with the binding of tritiated

acetazolamide as shown by several representative examples in Fig. 2.4. The effectiveness with which they compete with the

sulfonamide for the binding site follows the order of their potency as inhibitors of enzymatic activity, N3- < OCN- < HS- 5 CN-. Calculation of dissociation constants for the enzyme- anion complexes from the equilibrium data in Fig. 2A using the value of 8 X 10-S M for the enzyme-acetazolamide dissociation constant gives values of about 3.2 X 1OV M for N3-, about 3.2

II 011; I- I- -Am-- ”

1 i I IO+

I IO-’

I lO-6 IO+ lo-4

2 4 6 0 IO

PH FIG. 2. A, binding of SH-acetazolamicle to Zn(I1) carbonic

anhydrase B in the presence of metal-binding anions. Aliquots of human carbonic anhydrase B were equilibrated across Visking- N ojax membranes against the concentrations of 3H-acctazolamide given on the abscissa. After 48 hours, 0.5-ml samples nere re- moved and counted. Moles of 3H-acetazolamide bound per mole of protein are indicated on the ordinate. 0, no addition; V, 2 X 10-4~Na-;0,2X 10-4~OcN-;~,2X lo+~Hs-;A,2 X lo-’ M CN-; A, 0.5 M acetate. Protein concentrations were 2 X 10d6 M

for experiments below 10-e M acetazolamide, and 2 X 10e5 M for experiments above 10-G M acetazolamide. Values from 5 X 10-j M

to lo+ M acetazolamide were also checked with 1OV M protein. Conditions, 0.025 M Tris, pH 8.0, 4’. In the absence of anions, 0.5 mole of SH-acetazolamide is bound per mole of protein at 8 X 10-s M inhibitor indicating an equilibrium constant of 8 X 10-S M

for the complex. B, pH-dependence of 3H-acetazolamide binding to human carbonic anhydrase B. The equilibrium dialyses were carried out with 2 X 10-h M protein, 2 X 10-S M 3H-acetazolamide in 0.025 M Tris-O.OJ M sodium acetate, 4”. The sampling method was as described for A.

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X 1OV M for OCN-, and lop7 M or less for HS- and CN-, in close agreement with the Ki values which can be estimated from the concentrations of the anions required for 50% inhibition (31). The acetate anion, a product of the esterase reaction, is also effective in displacing the sulfonamide. Thus, in solution both the sulfonamide and the anionic inhibitors appear to share the common property of adding a coordinating ligand to the metal ion. In the recently reported crystal structure of the acetoxy- mercurisulfanilamide complex of human carbonic anhydrase C at 5.5A resolution, the sulfonamide group appears very near the zinc ion, probably close enough to be within the coordination sphere (32).

In addition to the competition between the anions and the sulfonamide, there are hydrogen ion equilibria which influence sulfonamide binding as shown by the pH-dependence of 3H- acetazolamide binding (Fig. 2B). The low dissociation constant for the Zn(I1) carbonic anhydrase-acetazolamide complex holds over a relatively narrow pH-range. Below pH 6.0 or above pH 8.0, the binding becomes rapidly weaker. By pH 4.5 or 10.0, only about 0.25 mole of acetazolamide is bound per mole of enzyme when free acetazolamide is equimolar to the enzyme at 2 X lop5 M. The pH range over which 1 mole is bound can be extended about a pH unit by increasing the free acetazolamide concentration. Practical limitations of the counting system, however, limit the free acetazolamide-enzyme ratio to about lO:l, so binding cannot be examined at high molar ratios of acetazolamide to enzyme.

Zinc content of human carbonic anhydrase B remains 1 g atom per mole over the pH range 4 to 10, as has been shown by the pH- dependence of 66Zn binding (12). Release of Zn(I1) at the extremes of pH is accompanied by radical alteration in protein conformation leading to much larger negative values of [a]238 (12, 33). Both of these changes occur well outside the pH range of acetazolamide binding and thus do not appear directly related to the factors controlling the pH-dependence of sulfona- mide binding.

The pH dependence of acetazolamide binding indicated by the equilibrium data presented here is similar to the pH-dependence reported for the binding of benzenesulfonamide to bovine carbonic anhydrase which was determined by a method which used inhibition kinetics (34). The inhibition constant of the latter sulfonamide, however, shows a maximum near pH 9, two pH units higher than the maximum equilibrium constant meas- ured here for acetazolamide. This difference may relate to the lower pK, of acetazolamide and would tend t.o confirm the sug- gestion of Kernohan (34) that the lower arm of the binding curve relates to the concentration of the anionic form of the inhibitor. Competition with -OH or some other ligand for the central metal ion may explain the loss of binding at high pH. The magnitude of the dissociation constant for the acetazolamide complex, 8 x 1OV M, however, would not indicate such sensitivity to pH if a metal-binding group alone were involved. Hence, there may be pH-induced changes in the protein molecule which influence the binding of parts of the sulfonamide not directly in contact with the cation.

Hydrogen Ion Equilibria Accompanying Reaction of Carbonic Anhydrase with Cyanide and SuZJide-If CN- and HS- inhibit carbonic anhydrase by coordinating the metal ion, the reaction should be accompanied by the displacement of a proton from the inhibitors at pH values below the pH region for the dissociation of HCN and H,S; the former described by a pK, of 9.3, the

I

PH

FIG. 3. Displacement of II+ and -01-I from human carbonic anhydrases B by cyanide and sulfide. The solid lines represent the continuolls titration curves for the dissociation of TICK to H+ and CN- and IIzS to II+ and -SH, expressed as the fraction of in- hibit.or undissociated (left ordinale). n t moles of II+ or -011 re- leased per mole of Zn(II) carbonic anhydrase WI the addition of equimolar sulfide. The remaining symbols refer to the moles of H+ or -OH released per mole of pro& on the addition of cyanide to Zn(I1) carbonic anhydrase (O), apocarbonic anbydrase (O), and Co(I1) carbonic anhydrase (0): Maxim~ml Iir and -OH release from the CoiII) ellzvme was not reached lultil the addition of about 1.5 eq of cyanide in agreement with the spectral data in Fig. 4. Temperature, 23”. ---, theoretical titration curve for an acidic group with a PK, of 8.1. expressed as moles of H+ dissoci- ated per mole-of protein (upper’ ri&(. ordinate). ~ - - and - - - --, tjheoretical difference titration curves expected for the displacement of H’~ (upper right ordim/e) and -011 (lower righl ordinate) from Zn(I1) carbonic anhydrase by cyanide and sulfide, respectively, as a function of pII, asstuning a single additional ionization (see the text).

latter by a pK, of 6.9 at about 23” (35). The net hydrogen ion release will depend on other hydrogen ion equilibria altered by the binding of the anions. In order to investigate the hydrogen ion equilibria that accompany the reaction of carbonic anhydrase with cyanide and sulfide, a set of equilibrium measurements bet,ween pH 6 and 10 were made by direct measurement of H+ and -OH release with the difference tit,ration method. The reaction of both cyanide and sulfide with carbonic anhydrase is characterized by a biphasic difference titration, H+ release at low pH followed by -OH release (H+ uptake) at high pH (Fig. 3). The entire reaction in each case is induced by the addition of 1 eq of inhibitor; excess up to IO-fold leads to no further reaction detectable by pH change. This finding is also in agreement with the stability constants for the cyanide and sulfide enzyme complexes (about 10m7 M at pH 8.0) calculated from the equilib- rium data (Fig. 2A) and from the Ki values derived from the concentrations required for 50% inhibition of CO2 hydration (31). These values predict full saturation of the Zn(I1) enzyme over the pH range 6 to 10 at the concentrations used (2 to 4 X 1OW M)

after the addition of 1 eq of inhibitor, assuming a zinc-anion bond as the major contributor to stability.

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5216 Mechanism of Action of Carbonic Anhydrase Vol. 242, X0. 22

A direct relat,ionship between the metal ion and the release of the H+ or -OH ion on reaction of the enzyme with cyanide is provided by the data on the apoenzyme. Neither proton nor hydroxide ion release occurs when cyanide is added to the apoenzyme (Fig. 3). A further connection between the difference titration and the catalytic mechanism is provided by the metal ion specificity for the H+ or -OH release. The biphasic cyanide titration curve, H+ release at low pH followed by -OH release at high pH, is limited to those metallocarbonic anhydrases, Zn(I1) and Co(II), which show significant catalytic activity (Fig. 3). Below pH 8.0, addition of 1 eq of cyanide to the other metal- locarbonic anhydrases of this series results in data like those shown for the apoenzyme. Around pH 9.0, the Ni(I1) and Cu(I1) enzymes actually show H+ release, as if the reaction were occurring accompanied only by the displacement of the proton from HCN.

The experimental curves can be theoretically calculated by utilizing the dissociations for the two inhibitors (solid lines, Fig. 3) and assuming a single additional hydrogen ion dissocia- tion associated with the protein and described by a pK, of -8 (dashed line, Fig. 3). Assuming that the anions are coordinating the metal ion, there are two possible interpretations for the H+ dissociation curve with a pK, of 8. The first alternative is to postulate that it represents the ionization of a coordinated Hz0 molecule with a pK, of 8. Participation of a coordinated -OH in the hydration reaction has been postulated by several investi- gators on the basis of kinetic studies (36, 37). Thus, at pH values at which the inhibitor dissociation curve is above the Hz0 dissociation curve, proto,ns should be released, and when it is below the Hz0 dissociation curve, hydroxide ions should be re- leased, as shown by the theoretical difference curves plotted for the two inhibitors. The observed dependence on the pK, of the inhibitor is predicted and the same additional dissociation curve serves to generate both the H2S and HCN difference titrations.

An alternate interpretation of the data can be made by con- structing a more complex model. The dissociation curve with an inflection at pH 8 (needed to generate the two difference titration curves) can be assumed to represent coordination to the metal ion of an additional protein ligand accompanied by displace- ment of a proton from this ligand. The displacement of this ligand by CN- at high pH could then be postulated to be ac- companied by the uptake of a proton, thus generating the upper limb of the difference titration. Coordination of an extra ligand to the cation at high pH has been postulated by Lindskog (31) and more recently by Dennard and Williams (38) on the basis of the spectral changes of the Co(I1) enzyme as a function of pH and by the observation that an additional H+ is released at pH 9 when Zn(I1) reacts with the apoenzyme (31). The appearance of a coordinated -OH was thought to be the less likely pos- sibility.

In light of the present data, several requirements of the latter alternative make it seem less attractive than the former interpre- tation. The cyanide titration in Fig. 3 requires the additional ligand group to have a pK, of 11 or above, since the intrinsic pK, of this group is not affecting the difference titration at pH 10. The pH of half-formation of the additional chelate ring would be about 8, therefore the affinity of the extra ligand for the cation must be such as to displace the dissociation of the hydrogen ion from the ligand at least 3 pH units lower. Assuming a pK, of 11 for the coordinating group, a log K, of 7 for the stepwise stability constant can be estimated from the data in Fig. 3. This

is a large value for a ligand so easily displaced from the cation by CN-, HS-, and a number of monodentate anions. Only 1 eq of cyanide or sulfide is required to displace it between pH 9 and 10 (Fig. 3). The contribution of such a ligand to the total stability of the complex at high pH would be even greater if conformations1 changes in the protein were invoked to explain the failure of this ligand to participate in the initial formation of the complex. Thus, the stability constant of the Zn(I1) enzyme should show a rapid increase between pH 7 and 9 with the addition of this ligand. The pH-dependence of the stability constant of the Zn (II) enzyme has been determined in detail (30) and no rapid in- crease is observed. The log of the stability constant rises linearly with pH from pH 5 to 10, at which value it becomes pH- independent.

The hydroxide ion interpretation does not involve these diffi- culties, since it requires only the ionization of a coordinated li- gand. The change from Hz0 to -OH would not be expected to change the stability of the complex significantly. The increasing concentration of the hydroxide form would also more adequately explain a variety of data which indicate competition between the metal-coordinating anions and some other competing group at high pH (31, 39) (see below). The biphasic difference titration is not readily accounted for on the basis of shifts in pK, values of neighboring protein groups. Such shifts would have to be induced differently by CN- and HS- or be variable with PH.

Formation of Co(IZ) Human Carbonic Anhydrase B-CN- Complex as Function of pll: Competition Between CN- and HC03--The visible absorption maxima of Co(I1) carbonic anhydrase associated with the d-d transitions of the metal ion have been studied in detail by several investigators (30, 31, 39). Changes in the energies and optical activity of these bands ac- companying sulfonamide and anion binding form much of the basis for the argument that these inhibitors occupy a position within the coordination sphere of the metal ion (12, 31, 39, 40). In connection with the difference titration of the Co(I1) enzyme, the spectra can be used to show two additional features of this system. As previously reported, there is a marked difference in visible absorption spectrum between the high and low pH forms of the enzyme (5, 31, 39) (Fig. 4A). Addition of CN-, however, generates an inhibitor complex with the same coordination geometry regardless of pH, since the spectrum of the complex is practically identical in the low, middle, and high pH range (Fig. 4B). At pH 6.0, 1.5 eq of CN- apparently leave the Co(I1) enzyme slightly undersaturated, in agreement with the complexo- metric titration which shows the release of only about 0.8 mole of H+ per mole of Co(I1) protein at pH 6.0. The spectral data in Fig. 4B thus support the idea, based on the dissociation constant of the enzyme-CN- complex, that CN- can be used to form the same complex between pH 6 and 10 without radical changes in coordination geometry.

In addition, the spectra can be used to show that both CN- and HC03 compete for the same binding site. At pH 8.2, over half of the enzyme is in the alkaline form (Fig. 3) and the 615 rnp and 640 rnp maxima are prominent (Fig. 4A). Addition of HC03 to this sample abolishes these maxima and gives a species similar to the protonated form of the enzyme at pH 6.0, as judged by the spectrum (Fig. 4C). The presence of HC03 interferes with the binding of CN-, since it now takes 3 eq of CN- to develop a spectrum comparable to that resulting from the addition of 1 to 2 eq in the absence of HC03-. The maximum peak heights in Figs. 4B and 40 are equivalent, since there is

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h.mw 10

FIG. 4. Visible absorption spectra of Co(D) carbonic anhy- drase. Complexes with -OH, CN-, and HCO,. A, -.-, 3.2 X 10e4 M Co(II) carbonic anhydrase, pH 9.0; --, 3.2 X lo+ M Co(II) carbonic anhydrase, pH 8.2; - - -, 3.0 X 1OW M Co(II) carbonic anhydrase, pH 6.0. B, (-.-) pH 9.0 sample from A plus 1.5 eq of cyanide, pH 9.0; -, pH 8.2 sample from A plus 1 and 2 eq of cyanide, pH 8.2; (- - -) pH 6.0 sample from A plus 1.5 eq of cya- nide, pH 6.0. C, --, pH 8.2 sample from A plus 0.5 M HCOa-, pH 3.2. D, -, sample from C plus 1, 2, 3, and 4 eq of cyanide, pH 8.2. The initial samples were those used for the complexo- metric titrations (Fig. 3) and were prepared as described under “Experimental Procedure.” The spectra are direct tracings from a Cary model 15 recording spectrophotometer and are uncorrected for scattering or trailing from the ultraviolet absorption. Full scale deflection was 0.1 O.D. unit, and the zero point was read- justed in midrun to record the spectra of the cyanide complexes.

Since the detailed investigation of the pH-rate profile for the human B isozyme revealed an inflection point near pH 8, con- siderably higher than pH 7.5, the highest value reported for the bovine enzyme (19), it seemed desirable to determine if the complexometric titration bore more than a fortuitous relation- ship to the pH-rate profile. Several isozyme and species variants were examined and it was found that inflection points of the esterase pH-rate profiles vary almost 1 pH unit as a function of species and isozyme (Fig. 6). Among the B isozymes, the mid- point varies from about 7.5 for bovine B to about 8.4 for the monkey B isozyme. In contrast, the midpoint of the monkey C isozyme shifts back to about 7.7, also characteristic of the human C isozyme. The cyanide complexometric titrations for the

I 2 Zn Co

I I I I

considerably more scattering in the sample to which solid bi-

carbonate has been added. Catalytic Activity as Function of pH: Relationship to the Cum-

pikrometric Titration-All pH-rate profiles published thus far for carbonic anhydrase are fairly adequately described by a sigmoid curve representing a single ionization. This appears to be true whether the substrate is COz (10, 39), an ester (19), or an alde- hyde (16). This pH-rate profile has been studied most ex- tensively for the bovine enzyme and has been interpreted to represent the pK, for a single group involved in catalysis (16, 19, 39). Detailed esterase pH-rate profiles for both the Zn(I1) and Co(I1) forms of the human isozyme B are shown in Fig. 5. Although esterase pH-rate profiles for individual preparations of the B enzyme follow relatively smooth curves, there is some variation in magnitude among different preparations, as indicated

6 f Q 4 lb PH

FIG. 5. Human carbonic anhydrase R. Correlation between the esterase pH-rate profiles of the Zn(II) and Co(I1) enzymes, t,he pH-dependence of the 640 nm absorption maximum of the Co(I1) enzyme, and the ionization curve derived from the com- plexometric titration. -, ionization curve with a pK, of 8.1 required to generate the difference titration curves in Fig. 3. l , esterase activity of Zn(I1) carbonic anhydrase assayed at 400 rnp; n , assayed at 348 rnp. A, esterase activity of Co(D) carbonic anhydrase assayed at 400 mr without excess cobalt in the reaction mixture; Cl, assayed at 343 rnp with a-fold molar excess cobalt in the reaction mixtures at pH 8.0 and above. All reaction mixtures above pH 9.0 contained 0.25 M Tris. 0, intensity of the 640 w maximum of Co(II) carbonic anhydrase expressed as A O.D.gro =

by the scatter in Fig. 5 showing the points for a number of O.D. (variable pH) - O.D. (pH 6.0). Conditions, 3 X 1OW M Co(I1) carbonic anhydrase, 0.025 M Tris, 1 X lo-’ M free Co(II), 23”.

preparations assayed at both 400 rnp and 348 m/l. Both curves can be fitted by a theoretical ionization curve with a pK, of 8.1, similar to the curve derived independently from the complex- ometric titration.

As previously shown for the bovine enzyme (31), the pH- dependence for the appearance of the 640 rnp absorption maxi- mum of the Co(D) enzyme follows a very similar curve (Fig. 5). Maximum intensity of this band for the human B enzyme is not reached until about pH 9.5. At the dilution required for assay, however, there is a precipitous drop in esterase activity of the cobalt enzyme above pH 9, typical of all cobalt isozymes, which apparently reflects the greater instability of the cobalt enzyme at the upper limits of the pH-stability curve. The presence of excess cobalt in the reaction mixture prevents this drop in activity. Substitution of Co(I1) results in a more active enzyme throughout the pH range of activity. Thus the differ- ence titration, the pH-dependence of the Co(I1) spectrum, and the pH-rate profiles for both active metals seem to reflect the same metal-coupled hydrogen ion dissociation.

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5218 Mechanism of Action of Carbonic Anhydrase Vol. 242, X0. 22

I-- 6 7

PH

FIG. 6. Complexometric titrat.ions and e&erase pa-rate profiles of carbonic anhydrase as a far&on of species and isozyme. Con- ditions were as described rmder “Experimental Procedure.” B, bovine enzyme B; 0, esterase activit,y; 0, cyanide difference titration; MB, monkey enzyme B; w, esterase activity; 0, cyanide difference titration; MC, monkey enzyme C; A, esterase activity. The solid ionization cnrves are drawn for pK, values of 7.5,7.7, and 8.4, respectively. The theoretical curves for the cyanide com- plexometric titrat.ions of the bovine B and monkey B enzymes were derived as in Fig. 3.

bovine and monkey isozymes B are also different and except for the most acid point in the case of the bovine enzyme, fit the theoretical curves derived as in Fig. 3 from the cyanide titration curve and the ionization curves fitted to the pH-rate profiles of the two isosymes (Fig. 6). Thus, it would appear that there is a relationship between the ionization detected by the complexo- metric titration and the pH-rate profile.

The complete titration curve for the human B enzyme reveals few dissociating groups from pH 6 to 10 (41,42), compatible with the observation that the protein shows little buffering capacity in this region and an ideal condit,ion for t.he complexometric titration. While a complete titration is not yet available for the bovine enzyme, the preparation exhaustively dialyzed against metal-free water requires considerably more acid or base to shift the pH in the range 6 to 7 than the human or monkey enzymes B. Thus, a larger number of dissociable hydrogens with pK, values in this region appear to be present in the bovine enzyme. This may interfere with the complexometric titration, although other factors such as changes in the stability of the enzyme or the cyanide complex could be responsible for the fall in hydrogen release in the acid region.

Suggested Mechanism of Action-If the ionization of a co- ordinated water molecule is the interpretation placed on the dissociation curve derived from the complexometric titration (Fig. 3), then the form of the enzyme active in the hydration and hydrolysis reactions is a mixed enzyme-Zn-hydroxide complex. This suggests a mechanism for the hydration reaction which is compatible with a large amount of other information presently available on carbonic anhydrase. The coordinated -OH may be visualized as attacking the COz carbon, Scheme 1, giving Inter- mediate A. There may, of course, be additional interactions contributing to the binding of CO*. Displacement of the inter-

mediate would then liberate bicarbonate regenerating the metal- hydroxide at high pH, favoring hydration and the hydrated species at low pH at which the dehydration reaction is known to proceed best (10). An analogous mechanism can be formulated for the hydrolysis of p-nitrophenyl acetate substituting an acetate intermediate. Both HC03 (Fig. 4) and acetate (Fig. 2A) can occupy the anion binding site. Replacement of a Zn(II)- acetate intermediate by water could be reversible and acetate is observed to be an inhibitor of the forward reaction.

A mechanism as formulated in Scheme 1 would explain the lack of inhibition by weakly binding anions at high pH (10, 39), the reversion at high pH of the spectra of the cobalt enzyme- anion or sulfonamide complexes to that typical of the alkaline form of the uninhibited enzyme (31,39), and the displacement of the pH-rate profile to higher pH in the presence of anions (10, 39). All can be related to competition with -OH, which at hieh enough concentration displaces the anions and generates the ac- tive enzyme. The above mechanism is compatible with the kinetic evidence which shows the anion binding site to be coupled to a group, the basic form of which is essential for the hydration of COz and the acidic form essential for the dehydration of bi- carbonate (10, 39).

The hydration reaction according to Scheme 1 is accompanied by proton transfers which are written in the simplest form. These transfers may be more complicated and be kinetically significant steps in catalysis. Imidazole or other adjacent pro- tein groups could participate in the hydrogen ion equilibria and influence the catalytic step. In light of present information, it does not appear necessary to postulate more than one group as responsible for the pH-rate profile. Packer and Meany (16) have suggested that the pH-rate profile is consistent with the participation of the unprotonated form of an imidazole group acting as a promoter for the direct transfer of water (formally -OH) from zinc to the substrate by extracting a proton from the coordinated water. However, there is an ionization associ- ated with the metal ion (postulated here to be that for a co- ordinated water molecule) with a pK, near neutrality and near the inflection point of the pH-rate profile (Figs. 5 and 6). Hence, a mechanism involving an unprotonated imidazole and a group coordinated to the metal ion must apparently include two ionizations. Complex mechanisms dependent upon differential reactivities of the various species resulting from the dissociation of two groups on a single molecule have been reported which produce only minor deviations from the sigmoid pH-rate profiles expected for a single ionization (43).

o=c=o t

&OH

-QH \ (A)

. -p

SCHEME 1

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Issue of November 25, 1967 J. E. Coleman 5219

While Scheme 1 is compatible with presently available data, it does not rule out other more complex mechanisms involving the participation of additional protein groups. If the alternate interpretation for the complexometric titration is made, mech- anisms involving the coordinated group in catalysis are con- siderably more complex. Dennard and Williams (38) have proposed a mechanism for the hydration reaction involving a co- ordinated group in the formation of a carbamic acid intermediate. Present information, however, does not favor these over the simpler mechanism involving a coordinated hydroxide.

Acknozuledgment-The excellent technical assistance of Mrs. Barbara Johnson is much appreciated.

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Joseph E. ColemanSULFONAMIDE, AND ANION BINDING

Mechanism of Action of Carbonic Anhydrase: SUBSTRATE,

1967, 242:5212-5219.J. Biol. Chem. 

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