Metal Binding and Catalytic Activity in Bovine Carbonic Anhydrase*

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 237, No. 4, April 1962

Printed in U.S.A

Metal Binding and Catalytic Activity in

Bovine Carbonic Anhydrase*

SVEN LINDSKOG AND Bo G. MALMSTR~~M

From the Enzyme Chemistry Group of the Institute of Biochemistry, University of Uppsala, Uppsala, Sweden

(Received for publication, October 2, 1961)

Carbonic anhydrase was the earliest known zinc metallo-enzyme (4). It has been isolated from various sources, mainly mammalian red blood cells (see (5)). Although several methods for the preparation of highly active enzyme were described earlier, adequate purification was not achieved nor was the stoichiometry of zinc established (5). Recently, bovine erythrocyte carbonic anhydrase, homogeneous by ultracentrifugal and ,electrophoretic analysis, was prepared in this laboratory (6).

The properties of this enzyme make it a feasible object for studies of the molecular basis of enzyme action. Thus, the enzyme can be obtained in large amounts; it is stable and contains 1 zinc ion as the only known nonprotein constituent; the molecular weight is relatively small, 31,000 (6) ; and convenient spectrophotometric assay methods can be employed (7). An investigation of the properties of the enzyme as a metal complex might give valuable information about its catalytic mechanism, particularly in combination with kinetic data and studies of the physical and chemical properties of the protein part. Such in- vestigations on several enzymes are in progress in this laboratory (cj. (2)), and the present paper is the first report on metal binding in carbonic anhydrase.

The metal is firmly bound, since it is not removed by extensive electrodialysis (8) and does not exchange with radioactive zinc at neutral pH (9). It has been found, however, that on dialysis against a chelating agent such as 1, IO-phenanthroline, at pH 5, the metal is dissociated with concomitant loss of enzyme activity (1). On addition of zinc to the zinc-depleted enzyme, the activity is completely regained (1).

Previous to this finding Vallee et al. (10) had demonstrated the parallel loss of metal and activity in another zinc metallo- enzyme, carboxypeptidase A, on dialysis against buffers of pH below 5.5 or at neutral pH if 1, lo-phenanthroline was included in the buffer. The metal ion specificity was not absolute, and several ions other than zinc were effective in restoring activity to metal-free carboxypeptidase (10, 11). Data providing strong evidence for the presence of a sulfhydryl group in the metal- binding site have recently been published (12, 13).

The results presented in this report show that in many respects carbonic anhydrase behaves similarly to carboxypeptidase.

* This work was supported by grants from the Swedish Natural Science Research Council and from the Division of General Medi- cal Sciences, United States Public Health Service (RG-6542). A preliminary account of part of this work has been published (1). Some of the data have been presented at the National Academy of Sciences Conference, Biological Aspects of Metal Binding, Uni- versity Park, Pennsylvania, 1960 (2) and the Fifth International Congress of Biochemistry, Moscow, 1961 (3).

Metal-free carbonic anhydrase has been prepared. It is comparatively stable, and no important change in particle size and secondary folding has taken place, as evidenced by ultracentrifu- gation and spectropolarimetry. Co* activates the metal-free enzyme, and Co*-carbonic anhydrase has been isolated. The most important difference is that no reactive -SH group is liberated on dissociation of the metal. Furthermore, methionine accounts for the entire sulfur content of the enzyme. These findings seem to exclude a zinc-sulfhydryl bond in bovine carbonic anhydrase.

EXPERIMENTAL PROCEDURE

Bovine erythrocyte carbonic anhydrase was prepared according to the method of Lindskog (6). The electrophoretic Component B (6) was used unless otherwise specified.

Chemicals-All solutions were made up with deionized water (14). Analytical grade salts were used without purification as the source of metal ions. Buffers were purified by dithizone extraction as described earlier (14). OP1 (Merck, analytical grade) and CMB (Sigma Chemical Company) were used without further purification.

Radioactive Zn-Zn65 with a specific activity of 1 mc per mg was obtained (Radiochemical Centre, Amersham, England) as a solution of zinc acetate. No other y-emitting isotope was detected by y-ray spectrometry, using a well-type scintillation counter (NaI(TI) crystal, Harshaw type 7F8; Photomultiplier, RCA 6342-A) coupled to a single-channel y-ray spectrometer (Baird Atomic 4522, model 810). In addition, no Cu (Cu is the stable disintegration product) was found by analysis which, with the quantity of sample used, means that the Cu:Zn ratio was <O.OOl; thus, any Cu contamination possibly present would, for

stoichiometric reasons, be negligible, independent of the binding strength of Cu*, since in all experiments with radioactive Zn the enzyme and metal are present in approximately equal molar concentrations. A radioactive stock solution was prepared by diluting 1.5 ml (containing 2.7 mg of Zn) of the original solution to 50 ml with 10e2 M inactive zinc acetate (the final Zn concentration being 1.1 X low2 M). The quantity of inactive Zn was chosen so that 1 ml of 10m4 M solution should give approximately 40,000 c.p.m. when the counting system was operated without any discrimination. By dilution of the stock solution, a series of l-ml samples of Zn solutions of known concentrations, usually in the range 10W5 to 10V4 M, was prepared. Concentrations of radioactive Zn were estimated by comparing the y-ray activity of l-ml samples with the standard series measured at the same

1 The abbreviation used is: OP, 1, lo-phenanthroline.

1129

by guest on January 13, 2019http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

1130 Metal Binding in Carbonic Anhydrase Vol. 237, No. 4

4 5 6 PH

FIG. 1. Equilibrium dialysis of carbonic anhydrase in the presence of OP and extraneous radioactive Zn, showing Zn* uptake in the protein compartment as a function of pH. Both cell compartments originally contained 0.1 M sodium acetate buffer, 4 X 10e5 M Zn*, and 1O-4 M OP. Enzyme concentration, 5.7 X lOwE M; liquid volume in each compartment, 2 ml; dialysis time, 13 days; temperature, 4”.

time, so that no correction for the decay of the isotope had to be evaluated.

Metal Analyses-Samples containing protein were treated with 10% trichloroacetic acid which has been found to liberate the metals quantitatively.z Zn was determined by the dithizone method (16) with the procedure described by Malmstrijm (14), except that a buffer of pH 5.5 and separatory funnels with Teflon stopcocks were used. Cu was determined spectrophotometrically at 620 rnp by the monocolor dithizone method (16); the samples, containing 1 to 2 pg of Cu, were extracted with 5 ml of dithizone solution (0.007 mg per ml). Co was determined spectrophotometrically at 510 rnp by the nitroso-R salt method (16), the final volume (5 ml) containing 1 to 10 pg of Co. In all cases, a series of standards was analyzed in parallel.

Protein concentrations were estimated spectrophotometrically at 280 mp. As determined previously, a 0.1 y0 solution of carbonic anydrase has an optical density of 1.80 at 280 rnp in a l- cm cell (6).

Sulfhydryl Groups-The spectrophotometric method according to Boyer (17), with CMB as a reagent, was employed. To 3 ml of a solution of metal-free carbonic anhydrase in buffer (0.6 mg of enzyme per ml = 2 X lop5 M), lo-p1 aliquots of 10e2 M CMB were added. The reaction was followed for several hours at 255 rnp in a l-cm cell with a Zeiss M4QII spectrophotometer. Blank experiments with all ingredients except enzyme were performed. A control experiment with a fresh solution of cysteine was also performed, and the sample of CMB used was found to react with an -SH-containing enzyme (muscle enolase).

Enzyme activity was determined by the calorimetric procedure described previously (6) except that a two-channel stopped-flow apparatus similar to that of Beers (18) was employed. OP at the final concentration of lop4 M was included in the assay mix- ture to remove traces of contaminating heavy metal ions3

2 B. G. Malmstrijm, unpublished experiments; see also (15). 3 Very small traces of ZnZ+ not removed by dithizone extraction

would give too high activity values when working with the metal- free enzyme, since this binds Zn2+ very strongly, and the maximal

Equilibrium dialysis technique according to Malmstriim (14) was used. In experiments with native carbonic anhydrase the enzyme was dialyzed overnight against 100 ml of 10m3 M OP at pH 7 to remove contaminating metal ions. Under these conditions the Zn content is not significantly changed (6). OP was finally removed by dialysis against deionized water.

Ultracentrifugations were performed at 20” and 59,780 r.p.m. with a Spinco model E ultracentrifuge. The photographs were evaluated and the sedimentation coefficients corrected for the influence of the viscosity and density of the medium by standard procedures (22).

Optical rotatory dispersion was measured with a recording spectropolarimeter manufactured by Rudolph Instruments Engineering Company. The experiments were performed at room temperature in a 5-cm cell with a protein concentration of 10 mg per m1.4

Electron spin resonance spectra were recorded with a Varian 4500 EPR spectrometer. The experiments were performed at 77” K according to a technique described previously (23). The spectral parameters were calculated5 as described (23).

RESULTS

Exchange Experiments-Carbonic anhydrase was dialyzed6 in equilibrium dialysis cells containing a series of 0.1 M sodium acetate buffers at pH values ranging from 3.4 to 5.5. The cells also contained lop4 M OP and 4 X 10e5 M Zn* 7 on both sides of the membrane. Measurements of the Zn* concentrations in both halves of the cells showed an uptake of Zn* with time in the enzyme-containing compartments; after approximately 2 weeks no further uptake was observed. In Fig. 1 the difference in Zn* concentrations on either side of the membrane at this time is plotted as a function of pH. For comparison similar experiments were carried out at pH 5 with bovine serum albumin, yeast enolase, and carboxypeptidase (Worthington), and in these experiments only carboxypeptidase took up Zn*. The results indicate that an exchange between enzyme-bound Zn and extraneous Zn* has occurred, the variation of Zn* uptake with pH reflecting a competition between ZnZ+ and H+ for the metal- binding site.

At pH 7.5 (0.1 M Tris-HCI buffer, 2 X low4 M OP, 4 X 10m5 M

Zn*, 4.8 X 10e5 M enzyme) a slow but measurable uptake occurred, although equilibrium was not attained within 1 month. Experiments were also carried out at pH 7.4 under conditions identical with those of Tupper, Watts, and Wormall (9), and, after a period of 51 days, resulted in a significant but very small Zn* binding corresponding to less than 5% of the total Zn content. It could not, however, be concluded that a true exchange had taken place, since carbonic anhydrase incubated with an excess of Zn* and immediately dialyzed against water still con-

enzyme concentration used was 5 X lo-* M. OP does not inhibit carbonic anhydrase (19,20). In fact, an activating effect has been reported (21), but in the present investigation a constant concentration of OP was used throughout.

4 The rotatory dispersion measurements were carried out by Dr. A. Rosenberg, and a detailed study will be published sep- arately.

5 The measurements and the calculations were kindly performed bv Dr. T. V&nn&rd. Institute of Phvsics. Universitv of Unnsala.

a 6 All experiments’ with proteins were ‘performed” in the cold (4-g”) unless otherwise specified.

7 Zn* symbolizes Zn containing Zne6, with the same specific radioactivity as the radioactive stock solution.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

April 1962 X. Lindskog and B. G. Malmstr~m 1131

tained small amounts of radioactivity even after a dialysis time of several days.

Another experiment (Fig. 2) illustrates the influence of urea on the chelating properties of carbonic anhydrase. The enzyme was dialyzed in a series of dialysis cells containing 0.1 M sodium acetate buffer, pH 5.0, 10e4 M OP, 4 X 10T5 M Zn*, and different concentrations of urea. With increasing urea concentration the amount of enzyme-bound Zn* decreased, suggesting a gradual destruction of the chelating site. No binding of Zn* to urea was observed at the concentrations used (2).

Reversible Dissociation of Zn-A 0.1 y0 enzyme solution was dialyzed against 100 ml of 0.1 M sodium acetate buffer, pH 5.0, containing also 10M3 M OP. At measured intervals aliquots were withdrawn and the remaining activity was determined. To part of the withdrawn samples (containing OP) Zn* was added, to a final concentration of 2 X 10e3 M, and the activity was measured again. The results are shown in Fig. 3 for two different samples of carbonic anhydrase. The activity slowly decreased and 50% of the original value was reached after approximately 40 hours and 10 days, respectively. Carbonic anhydrase exists in two forms, separable by zone electrophoresis (6), and, in all instances investigated, the “fast” loss of activity occurred with the electrophoretic Component B while the il component showed a “slow” behavior. In both cases addition of Zn* resulted in a complete recovery of enzyme activity as compared to a control consisting of enzyme dialyzed against the same buffer without OP. The reactivation cannot be due to removal of OP, which acts as an inhibitor, since other metal ions, which under the conditions used do not inhibit the enzyme, fail to reactivate it (1).

A sample dialyzed as described above, with a residual activity of 1% of the native enzyme, was analyzed for Zn after removal of OP by dialysis against deionized water. No Zn was observed although such an amount of enzyme was analyzed that 1 y0 of the original Zn content should have been detected.

A dialysis experiment, performed at pH 4.0 and the same total OP concentration as above (1O-3 M), did not result in an appreciably faster loss of activity, and to a considerable extent the inactivation was irreversible. Carbonic anhydrase dialyzed against 0.1 M Tris-HCl buffer, pH 7.3, containing 5 X 10-s M OP, only lost 30% of its activity in 16 days.

Increased OP concentrations resulted in accelerated dissociation, and in order to prepare larger amounts of metal-free enzyme the following procedure has been employed: 100 mg of freeze-dried carbonic anhydrase are dissolved in 3 ml of 0.1 M sodium acetate buffer, pH 5.0, containing 10-Z M OP, and dialyzed against 300 ml of the same solution, until the residual activity is less than 3 ‘%, which is reached in approximately 2 and 7 days with Components B and A, respectively. Finally, OP is removed by dialysis against frequent changes of deionized water or metal-free buffer, and the small precipitate, usually formed, is removed by centrifugation.

By addition of small portions of Zn2+ to metal-free enzyme the activity is restored in direct proportion to the total amount of Zn*, as illustrated in Fig. 4. The activity is not further increased when Zn* corresponding to 1 g ion per mole of enzyme has been added.

The close correlation between Z& and activity is also demonstrated in an experiment, showing that the activity is proportional to the amount of enzyme containing strongly bound Zn*. Radioactively labeled carbonic anhydrase was prepared by reactivation of metal-free enzyme with Zn*. After removal of

0 I 0 2 4 6 8

Urea cont. (u)

FIG. 2. Equilibrium dialysis of carbonic anhydrase (CA) in the presence of OP, extraneous radioactive Zn, and urea, showing Zn* uptake in the protein compartment as a function of urea concentration. Both cell compartments originally contained 0.1 M sodium acetate buffer, pH 5.0,4 X 10m6 M Zn*, lo-* M OP, and urea. Enzyme concentration, 8.7 X lo+ M; liquid volume in each compartment, 2 ml; dialysis time, 22 days; temperature, 4’.

. .

< 91

’ 25

0 0 5 10 15 20

Time (days)

FIG. 3. Loss and recovery of carbonic anhydrase activity during dialysis against OP. Three milliliters of a 0.1% (3 X 10m6 M) enzyme solution dialyzed at 4” against 100 ml of 0.1 M sodium acetate buffer, pH 5.0, containing 1OF M OP, with change of dialysis liquid every 48 hours. A, Component A; 0, Component B. To 100-J aliquots, containing OP, 25 kl of 1OW M Zn2+ were added and the activity was measured. A, Component A; 0, Component B; both after correction for dilution.

loosely bound Zn* by dialysis against 10m3 M OP at pH 7 for 15 hours, the enzyme contained radioactivity corresponding to 1.0 Zn* per molecule. Furt,her dialysis against lop3 M OP at pH 5.0 resulted in a parallel loss of Zn* and enzyme activity as shown in Fig. 5.

Some Properties of Metal-free Carbonic Anhydrase-The metal- free enzyme, prepared as described in the previous section, is comparatively stable in water and in buffers in the pH region 5 to 9. On standing for more than a few weeks at pH 5 the potential activity slowly decreases and a precipitate is formed. An aqueous solution of the metal-free enzyme, which had been left in the refrigerator for 6 weeks, could be reactivated to 70% of the original value, whereas the native enzyme loses only approximately 10 ‘j& activity under the same conditions.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


The readily effected reactivation and the stability indicate that no appreciable change of the molecular structure of the

enzyme has occurred on dissociation of the metal. In order to

investigate this, ultracentrifugations were performed at two pH

values, where the metal-free enzyme is stable. The enzyme concentration in both experiments was 10 mg per ml, and the buffers used were 0.1 M sodium phosphate buffer, pH 7.5, and

0.1 M sodium acetate buffer, pH 5.0, both containing 10-d M OP to prevent the metal-free enzyme from combining with metal

100 c

0 , 0 0.25 0.5 0.75 1.0 2.0 3.0 Molar ratio added Zn’+/meial-free CA

I I I I I

FIG. 4. Restoration of activity to metal-free carbonic anhydrase (CA) in relation to the amount of added Zn*+. To 3 ml of metal-free enzyme (2.2 X lo+ M; residual activity, 3’%) at 22”, aliquots of 1.1 X 10W2 M Zn2+ were added.

100 I I I

I I I

0 5 IO r5 20

Time (days)

FIG. 5. Loss of Zn and carbonic anhydrase activity during dialysis against OP. Two milliliters of a 3 X 1OV M solution of radioactive enzyme (Component A) (9000 counts min-1 ml-l) dialyzed under the same conditions as in Fig. 3. 0, Relative enzyme activity; 0, relative radioactivity.

TABLE I

Some properties of Zn-free carbonic anhydrase in comparison to native enzyme

Property I Zn-free I Native / !

Zn content. ................ Relative activity. ..........

sg, ........................ [LYL .........................

x, ..........................

< 0.002% 0.21% 1 100 2.8 s 2.8 S

-60” -60” 226 mp 226 rnp

TABLE II

Determination of apparent dissociation constants for Znzf-carbonic anhydrase at pH 5.0 and Y.0

OP, 3.0 X 10e4 M, approximately 4 X 10e5 M Zn*, and 0.1 M

sodium acetate buffer, pH 5.0, or 0.1 M sodium phosphate buffer, pH 7.0, were present in both cell compartments. The concentration of Zn-free enzyme was 4.7 X 10-j M. The radioactivity was

measured after 7 days at 25”.

Parameter

Zn* concentration

pH 5.0 pH 7.0

a. Protein compartment. 6.4 X 10m5 M 6.0 X 1o-6 M

b. Nonprotein compartment 3.4 X lo+ M 1.7 x 1o-5 M

Y............................. 0.64 0.91 [Zn2+]free t. 2.5 X IOWO M 1.0 x lo-” M

pKd.......................... 9.9 12.0

t Calculated, with constants given in (25).

ion contaminations possibly present in the ultracentrifuge cells. After the run, residual activity was measured and no significant increase was observed. Further, the potency of reactivation had not been impaired. In both instances metal-free carbonic anhydrase gave a single symmetrical peak, indistinguishable from that of the native enzyme.

In addition, the optical rotation was investigated in the wave length region 700 mp to 310 rnp at an enzyme concentration of 10 mg per ml (0.1 M sodium acetate buffer, pH 5.0). The curves obtained for the metal-free and native enzyme were identical within the experimental error, showing that no gross change of the protein conformation had taken place. [a], and X, were calculated according to Lowry (24), with the method of least squares. The above results are summarized in Table I.

Apparent Dissociation Constant of ZrP-Carbonic Anhydrase-

Since the dissociation of Zn2+ has been shown to be reversible, a quantitative estimation of the binding strength should be feasible. The metal dissociates only when the free metal ion concentration is sufficiently low, at a level which has been attained by including OP in the system. Metal-free enzyme was dialyzed at 25” in equilibrium dialysis cells containing a series of buffers of pH values from 4.4 to 7.5. The buffers also contained Zn* and OP at such concentrations that only a fraction (P) of the enzyme contained Zn* at equilibrium (dialysis time, 7 to 8 days). The ti values were estimated as the difference in the Zn* concentrations for the two cell compartments divided by the molar enzyme concentration.* On the assumption that Donnan effects were negligible (cf. (14)), concentrations of free Zn* were calculated from the measured total Zn* concentrations in the nonprotein compartments, using the values for the acid dissociation constant of OP.H+ and the stepwise stability constants of the Zn-OP complexes as determined by Kolthoff, Leussing, and Lee

(25). Finally, the apparent dissociation constants, Kd, were calculated with the formula: & = [Zn*],,,.(l - ij)/s;, where KW+lfree represents the calculated concentration of free hydrated

8 Too low values of t were occasionally obtained which were due to the presence of denatured material. The molar concentration of metal-free carbonic anhydrase capable of binding metal was estimated in a control equilibrium dialysis cell, containing 0.1 M

sodium acetate buffer (without OP) and Zn*. The amount of Zn bound was assumed to represent tightly bound metal in view of the low pH and free Zn2+ concentration (5.0 and approximately 1OW M, respectively).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

April 1962 X. Lindskog and B. G. Malmstriim 1133

Xnzc. The procedure is illustrated in Table II, where further experimental details are given. Kd changes by a factor of lo4 to lo5 in the pH region investigated as shown in Fig. 6, where p& is plotted as a function of pH; the values have not been corrected for complex formation with the buffer anions.

TABLE III Evidence for absence of cysteine at metal-binding site of

bovine carbonic anhydrase

Experimental criterion No. of residues or groups per mole of enzyme

In a similar experiment the electrophoretic Components A and B were compared under identical conditions (pH 5.0). After a period of 8 days the B values were identica.1 and remained constant during the time of observation (20 days). However, at the end of this period a slight precipitate was observed, so that the true equilibrium concentrations may not have been obtained for the “slow” A component.

Metal Ion XpeciJicity-Metal ions were added (lo-’ M) to 50-~1 samples of metal-free enzyme in sodium acetate buffer, pH 5.0. The enzyme concentration was 1.0 mg per ml (3 X 10e5 M) with a residual activity of 4% of the native enzyme. Aliquots of 10 ~1 of the mixtures were pipetted into 5 ml of assay medium, and the enzyme activity was estimated. Of the ions investigated Co2+ was the most efficient in restoring activity. In contrast to the reactiva.tion by Z&, where no time lag has been observed, the Coz+ activation required several hours as shown in Fig. 7. By increasing the temperature or Co* concentration, the activation was accelerated, but the same final activity (45 f 5% of the value for the native enzyme) was always obtained.

Weak effects of Ni2+ and Fe* have been found (3), which have the same order of magnitude as the residual activity, but these observations have not been fully reproducible and might be results of Zn* contamination. No activation was obtained with CL@, Cd*, Pb*, HgN, Be*, and Fe3+. The following ions were investigated at a total final concentration of 10m3 M:

Mg*, Cazf, Bati, and Mn*, but no effect was observed for any of these divalent ions.

Some Properties of Co-Carbonic Anhydrase-In order to prepare Co-carbonic anhydrase an aqueous solution of metal-free enzyme (5 x 10e4 M) was incubated with 10e3 M Co*. Gradually a reddish blue color developed. After 20 hours loosely bound Co* was removed by dialysis against deionized water. The specific activity of the product was the same as the maximal value obtained in the CoLy- activation experiments described in the preceding section. A dialysis experiment with radioactive co* and Zn-free enzyme showed a binding of 0.95 g ion per mole of protein in 0.1 M acetate buffer, pH 5.0 (the concentration of free Co* at equilibrium was 1.3 X 1O-5 M).

I f a solution of Co-carbonic anyhdrase is incubated with Zn*, Co2+ is gradually displaced with a concomitant increase of activity from the value for Co-carbonic anhydrase to that of the native enzyme. Experiments were performed with radioactive Zn2f in a series of equilibrium dialysis cells. At intervals the contents of both cell halves were removed and analyzed for Zn* and Co. The results and detailed experimental conditions are described in Fig. 8 and its caption. During the course of the displacement the sum of enzyme-bound Co* and Zn* remained close to 1 g ion per mole, the maximal deviation being 0.14 g ion per mole. The measured enzyme activities closely corresponded to the values calculated from the observed proportion of bound Co* and Zn*. When the native enzyme was incubated with a IO-fold excess of Co2+, no displacement was observed. These results indicate that both metals are bound to the same enzymic site, which has a smaller affinity for Co* than for Zn2+.

The characteristic visible absorption spectrum of Co-carbonic

CMB reaction a. pH 7.0 ...................... b. pH 5.0 ...................... c. pH5.0,6Murea.. ..........

Cysteic acid ..................... Methionine sulfone ............... Total S ..........................

<0.07 <0.02 <0.07

0.2 f 0.1* 3.0 f 0.2

. 3.2 f 0.1

* Average of five determinations on separate preparations; two fully active preparations showed <O.l residue per mole.

p%’

4 5 6 7 8 PH

FIG. 6. Negative logarithms of the apparent dissociation constants of Zn2+-carbonic anhydrase as a function of pH. l ,O.l M sodium acetate buffers; 0, 0.1 M sodium phosphate buffers. For further details, see text and Table II.

‘“l-----l’ 940-

/--- _*c--

: 1 --

2 g so-

<

d 20-

10

Time [hours)

FIG. 7. Time course for the Co2+ activation of metal-free carbonic anhvdrase. Enzvme concentration. 2.2 X 1OW M: Co2+ was added and aliquots withdrawn for activity measurements. 0, 4 x 1o-4 M co2+, 4’; A, 4 x 1O-4 M CO'+, 22"; 0, 2 x 1O-3 M

co2+, 22".

anhydrase is shown in Fig. 9. The wave length of maximal absorption is 553 f 2 rnp, i.e. a red-shift (compare free hydrated Co*). Three “shoulders” are situated at approximately 585 rnp, 610 mp, and 630 rnp, respectively. The molar extinction


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


coefficient, E, at the maximum is 290 f 20 liter mole-l cm-l, larger by a factor of approximately 30 than that of the free Co* ion.

Some Properties of Cu-carbonic Anhydrase-Addition of stoichiometric amounts of Cuzt to the metal-free enzyme gives an enzymically inactive compound, and activity is not restored on subsequent addition of Znzf. On removal of CL@- by dialysis against OP-containing buffer at pH 5.0, enzymic activity can again be restored, indicating identical binding sites for the 2 metal ions rather than denaturation.

A concentrated solution of Cu-carbonic anhydrase (~10 mg per ml) has a visible greenish color. The absorption spectrum shows a broad band with a maximum at 760 f 10 rnp, and with a molar extinction coefficient of 120 f 10 liter mole-l cm-l (Fig. 9).

I I

1. 0 100 .

Time (days)

FIG. 8. The displacement of Co2f by radioactive Zn2f from Co2+-carbonic anhvdrase in 0.1 M sodium acetate buffer. nH 5.0. at 4”. Enzyme concentration, 5.0 X 1O-5 M; initial Zn* concen: tration, 9 X 10e6 M; final free Zn2+ concentration, 6.7 X lop6 M;

final free Co2+ concentration, 4.4 X lo+ M. i represents g ions of metal bound per mole of protein. A, Co2+; 0, Zn2+; 0, activity expressed as per cent of activity of Zn2+-carbonic anhydrase. The curve (- - - -) is calculated from the observed proportions of enzyme-bound Co2+ and Zn2+.

3oc

20(

&

10(

I I I I I

/ I / \I I I 400 500 600 700 800 900 1

wavelength (m,u]

. 1 00 0

FIG. 9. Absorption spectra of Coz+- and Cu2+-carbonic anhydrase (CA) measured at 22” in a Zeiss PRQ20A recording spectrophotometer with native enzyme at the same concentration in the reference cell. The absorption was measured on 4.7 X lo-* M and 3.3 X 1OF M enzyme solutions (pH 5.8), respectively, but the values have been converted to molar extinction coefficients (c, liter mole-l cm-l).

Cu-carbonic anhydrase is paramagnetic and a frozen aqueous solution at 77” K gives an electron spin resonance spectrum of the asymmetric structure characteristic for CL?+ complexes (cf. (23)). When the same treatment as described previously was applied (23), the following parameters were calculated: gm = 2.077, gli = 2.310, 1 A 1 = 0.0133 cm-l, cr2 = 0.61, and (4/7 aI2 + K) = 0.72.

Evidence for Absence of Cysteine at Chelating Site-The recent finding by Vallee et al. (12) that one reactive thiol group is liberated on dissociation of the metal in carboxypeptidase raised the question whether coordination to -SH is present in other zinc metallo-enzymes as well. With metal-free bovine carbonic anhydrase, however, no significant reaction with CMB has been observed. Experiments were performed in 0.1 M sodium acetate buffer, pH 5.0 (both without and in the presence of 6 M urea), and in 0.1 M sodium phosphate buffer, pH 6.8. Under none of these conditions was more than 0.1 -SH group per mole of enzyme observed. Potential enzyme activity was not significantly decreased by CMB.

Furthermore, amino acid analysis, according to the Moore- Stein technique: of hydrolysates of performic acid-oxidized carbonic anhydrase gave only nonstoichiometric amounts of cysteic acid.lO The content of methionine sulfone closely corresponded to the total sulfur content as observed by independent analysis (6) .ll Further details are given in Table III.

DISCUSSION

Earlier data on the zinc content of carbonic anhydrase varied appreciably (27), leaving the question of the stoichiometry of zinc unanswered. The present investigation has been performed with carbonic anhydrase prepared from bovine erythrocytes and purified by chromatography and zone electrophoresis. The purified enzyme is homogeneous in electrophoresis and in the ultracentrifuge as has been shown previously (6). The zinc content is 0.21%, corresponding to 1.0 g atom of Zn per molecular weight, 31,000 (6). The enzyme conforms to the criteria (28) of a metallo-enzyme (4, 6), and the close correlation between Zn content and carbonic anhydrase activity of erythrocytes (29) supports the reasonable assumption that the enzyme contains Zn in vivo.

A striking demonstration of the firmness of the attachment of Zn was given by Tupper, Watts, and Wormall (9) who showed that the metal did not exchange with radioactive Zn2+ at neutral pH even during a period of 32 days. These results have largely been confirmed here; possibly a small exchange occurs after an incubation period of 51 days.

If the free metal ion concentration is considerably lowered by introducing a chelating agent, such as OP, into the system, radioactive Zn can be taken up by the enzyme (Fig. 1). Neither serum albumin, which binds Z& only by simple complex formation (30), nor enolase, a Zn2+-activated enzyme with a specific

9 A detailed amino acid analysis of bovine carbonic anhydrase has been performed and will be published elsewhere.

‘0 We are indebted to Dr. Stanford Moore for surveying our analytical data on performic acid-oxidized carbonic anhydrase and for instructing us, during his recent visit to Uppsala, in the use of a modified oxidation method (S. Moore, unpublished).

I1 With the analytical method used (26) the maximal deviation in the analysis of proteins has been estimated to be approximately 1 fig of S. Since samples containing about 25 pg of S were analyzed, this corresponds to an uncertainty of CO.2 residue per mole of enzyme.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

April 1962 S. Lindskog and B. G. Malmstriim 1135

binding site (2, 31), bound zinc under the experimental conditions employed, whereas carboxypeptidase, a zinc metallo- enzyme (28)) behaved analogously to carbonic anhydrase. These results indicate that an exchange with the tightly enzyme-bound Zns has occurred. Since complete metal analysis of carbonic anhydrase has not been performed (the enzyme has been shown to be free of Cu and Fe; cf. (6)), the possibility exists that the radioactive Zn was bound to another site with strongly chelating properties, originally containing some metal other than Zn. This is not very probable, because the zinc-free enzyme, prepared by dialysis against still higher concentrations of OP, can only bind 1 Zn* per molecule strongly.

At pH 7.8 the Zn* uptake is extremely slow, but the rate in- creases when pH is lowered, equilibrium being reached after approximately 2 weeks at pH 5. The amount of Zn* bound at constant total OP concentration rapidly decreases below pH 5 (Fig. l), reflecting a weakened binding strength, as expected, since the presumptive ligands provided by the protein are polar groups also capable of binding protons (cj. (32)). It has been shown by Keller (33) that, at pH 2.9, exchange with radioactive Zn takes place even in the absence of a chelating agent. At this pH the enzyme is inactivated, but activity is partly restored on neutralization (34).

It has been pointed out (35, 36) that the chelating properties of a protein should disappear on denaturation, and this has been demonstrated for conalbumin and yeast enolase (2). The experiment shown in Fig. 2 illustrates qualitatively that at high concentrations of urea the Zn* binding is considerably weakened. Quantitative estimates of the effects of urea on the metal affinity, enzyme activity, and protein conformation are now in progress.

Since previous attempts to remove the metal caused irreversible inactivation (34), earlier evidence for the assumption that the metal is necessary for the catalytic activity of carbonic anhydrase was indirect, being based on inhibitor studies. Thus, certain anions, such as cyanide, azide and sulfide, which bind strongly to Zn*, are powerful inhibitors (cf. (7)). The finding that Zn can be removed by dialysis against OP at pH 5, and the demonstration of a close correlation between zinc content and activity (Figs. 4 and 5), comprise new and strong evidence for the functional role of Zn in the active site of carbonic anhydrase.

Zn is also an essential constituent of carboxypeptidase as shown by Vallee et al. (lo), and a comparison between the two metallo-enzymes should be of interest. The metal is easily dissociated from carboxypeptidase by dialysis against OP at neutral pH or below pH 5.5 even in the absence of OP (10); this indicates a weaker binding than in carbonic anhydrase. The apparent dissociation constant for Zn-carboxypeptidase at pH 8 is 3 X lo-l1 M (13), whereas under similar conditions (pH 7.5) the corresponding value for Zn*-carbonic anhydrase is approximately lo-12 M (Fig. 6). Preliminary equilibrium dialysis experiments carried out at this laboratory (1) indicated a similar binding strength for the two enzymes. The enzymes were dialyzed against a buffer, pH 5, containing 1 M NaCl, OP, and extraneous Zn*. The radioactive Zn exchanged with the enzyme-bound metal, and the final degree of binding was calculated assuming complete equilibrium. For carbonic anhydrase, this was probably not the case. As seen in Fig. 3, the

l2 R. C. Warner, unpublished experiments quoted in (35).

rate of dissociation is slow and the calculated binding was presumably too low. The same binding strength has been found for the two electrophoretic components of carbonic anhydrase, but this might be artificial because of the extremely slow dissociation rate for Component A (Fig. 3). Differences in the rates of dissociation have also been observed for the separate forms of human erythrocyte carbonic anhydrase. This enzyme can be separated by chromatography or electrophoresis into components with different specific activities (37, 38).13 The low activity form loses zinc relatively rapidly ((38) ; see also Fig. 9 in (2)), whereas the high activity enzyme shows a dissociation rate at least as low as that of Component A in the bovine enzyme (2). The fact that OP and other chelating agents, which inhibit carboxypeptidase and other zinc metallo-enzymes (27)) are not carbonic anhydrase inhibitors suggests that the metal is inaccessible to these reagents and strengthens the assumption (2) that the zinc ion is “buried” in the protein structure, e.g. by having the four strong coordination sites of a tetrahedron occupied by ligands contributed by the protein, as proposed by Williams (39). In addition to steric effects, the possibility of the electrostatic charge surrounding the metal ion influencing the dissociation rate has been discussed (2).

The metal ion is not essential for keeping the enzyme molecule in its specific conformation, as in carnosinase (40), or for hold- ing separate peptide chains together, as in alcohol dehydrogenase (41). Thus, in analogy to carboxypeptidase (42), the metal-free enzyme has physical properties similar to those of the holo- enzyme (Table I). This, of course, does not imply that the metal ion does not affect enzyme stability at all. Zn*-carbonic anhydrase is fully inactivated in the presence of 4 M urea, but the activity is completely recovered on dilution of the urea14 (cf. (20)). Metal-free carbonic anhydrase in 4 M urea solution, however, is not reactivated after addition of Zn* and immediate dilution. On standing with Zn*, the reversibility is gradually regained, being complete after a period of several hours.” These phenomena are being further investigated.

Although Zn* probably is the “natural” metal ion in carbonic anhydrase, Co* is also effective in restoring activity to the apoenzyme. For carboxypeptidase, Co2+ is likewise an efficient coenzyme but the complexes with Ni2+, Fe&, and Mn* also give reasonably high activities. The metal specificity of carbonic anhydrase seems to be narrower; only very small effects of Ni* and Fe* have been observed and cannot be considered to be fully established. Of course, it cannot be excluded that these and other ions might activate, since, apart from Zn-carbonic anhydrase, only the Co and Cu enzymes have actually been isolated and tested for enzymic activity. It is possible that an ion with a relatively low affinity is capable of activation. The principal difference between a metallo-enzyme and a metal- activated enzyme, which is often only a matter of binding strength (27, 43), would thus become strikingly small. In further experiments, the conditions will be extended to a larger range of metal ion concentrations, combining metal binding and activation studies.

Activation by Co2+ is time-dependent (Fig. 7), possibly re- quiring a rearrangement of the chelating site, but in the end a reddish blue complex is formed containing 1 tightly bound Co* ion per molecule and with 45% of the activity of the

I3 We wish to thank Drs. E. E. Rickli and J. T. Edsall for mak- ing the manuscript available before publication.

I4 S. Lindskog, unpublished experiments.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


native enzyme. That this metal binds to the same site as Zn* is evidenced in the experiment illustrated in Fig. 8. The results also indicate that Co* is bound more weakly than Zn*.

Very little can be concluded about the mode of coordination of Zn2+ in carbonic anhydrase. The dependence of the apparent dissociation constant on pH (Fig. 6, cf. also Fig. 1) gives no direct clue about the pK, values of the polar groups involved. The parameters calculated from the electron spin resonance spectrum of Cu-carbonic anhydrase are not strikingly similar to any Cu* complex previously investigated (23), and too little is known about the relation between such spectral parameters and the structure of Cu* complexes to permit any conclusion as to the nature of the ligands. ’

In carboxypeptidase one sulfhydryl group becomes exposed on removal of the metal (12), and, by comparing stability constants for a number of metallocarboxypeptidases and for various model complexes, strong evidence has been obtained for the participation of -SH and N (imidazole or amino group) as ligands in this enzyme. Williams (44) has pointed out that if the Zn2+ complex is more stable than the Ni* and Co& complexes, and if the spectrum of the Co* complex shows a shift of the absorption maximum to a longer wave length (compared to the free metal ion) in combination with an increased specific absorption, the metal is probably coordinated to S. This is the case for carboxypeptidase, and carbonic anhydrase shows the same properties. In fact, Keller, Gottwald, and Wendling (45) have reported that two reactive SH groups are liberated on acid inactivation of carbonic anhydrase. After neutralization, activity is gradually recovered and the -SH groups become concomitantly unreactive. Further, Rickli and Edsall (38) recently reported evidence for one -SH group being involved in binding the Zn of the low active form of human carbonic anhydrase.16 However, the evidence presented in Table III, showing the absence of stoichiometric amounts of cysteine and a methionine content corresponding closely to the total sulfur, seems to exclude the involvement of an -SH group in Zn binding in bovine carbonic anhydrase.

Recently it has been found that, in addition to several anions, some Zn* (39) and Co* model complexes catalyze the reaction: COP + Hz0 ti H&0z.‘6 The active complexes are all tetra- hedral, octahedral complexes being inactive, and Williams has pointed out16 that the spectra of Co- and Cu-carbonic anhydrase may also be explained by assuming a highly asymmetric coordination with a ligand number less than 6 (presumably 4), not necessarily comprising -SH. It is hoped that further investi- gations on the properties of metallocarbonic anhydrases and model complexes will yield more definite information about the mode of metal binding in this enzyme.

SUMMARY

The interactions of bovine carbonic anhydrase with zinc and some other metal ions have been studied. The enzyme, containing 1 zinc ion per molecule, loses activity on dialysis at pH 5 against 10e3 M 1, lo-phenanthroline; concomitantly the metal is dissociated. The dissociation is slow, the two electrophoretic forms of the enzyme showing different rates; half the activity

15 In agreement with the results reported here, these investi- gators found no -SH groups in bovine carbonic anhydrase with the CMB method (E. E. Rickli and J. T. Edsall, personal communication).

16 R. J. P. Williams, personal communication.

is lost in approximately 40 hours and 10 days, respectively. On readdition of Zn*, activity is completely regained. Activity loss and recovery have been shown to be directly proportional to the zinc content. The apparent dissociation constant for Zn*-carbonic anhydrase is lo-l2 M at pH 7.5. The binding becomes weaker at lower pH values, reflecting competition by protons for the chelating site. A denaturing agent, such as urea, also impairs the zinc affinity.

Metal-free carbonic anhydrase has been prepared and has a sedimentation coefficient and rotatory dispersion indistinguishable from the native enzyme, indicating that no gross change of the protein structure has occurred.

Of a number of metal ions tested, Co2+ was effective in restoring activity to the metal-free enzyme. Weak effects of Ni* and Fe* have also been observed. The Co*-enzyme has been isolated. It has 45% of the activity of the native enzyme, and has a visible absorption spectrum with a maximum at 553 rnp and a specific absorption (E,,,) of 290 =t 20 liter mole-1 cm-l. Evidence has been presented for Co* and Zn* binding to the same enzymic site, Co* being more weakly bound than Zn2+. Cu2+-carbonic anhydrase is inactive; the absorption spectrum has a maximum at 760 mp (emax = 120 f 10 liter mole-l cm-l). The electron spin resonance spectrum of the complex is analo- gous to other Cu (II) complexes.

Metal-free carbonic anhydrase does not react with p-mercuri- benzoate under various conditions, and amino acid analyses have demonstrated the absence of stoichiometric amounts of half- cystine in the enzyme. The present data give no direct clue to the mode of metal binding in bovine carbonic anhydrase but are in accord with the metal being coordinated to less than six (possibly four) ligands, the nature of which is as yet unknown.

Acknowledgments-We wish to thank Miss Barbro Kumlin, Mr. S. 0. Falkbring, and Mr. S. Bergvall for skillful technical assistance.

REFERENCES

1. LINDSKOG,~., AND MALMSTR~~M, B. G., Biochem.Biophys. Re- search Communs., 2, 213 (1960).

2. MALMSTR~~M, B. G., Federation Proc., 20 (suppl. lo), 60 (1961). 3. MALMSTR~M, B.G., ROSENBERG, A., AND LINDSKOG,~., in P.

DESNUELLE AND A. E. BRAUNSTEIN (Editors), Symposium IV of thejifth international congress of biochemistry, Moscow, 1961, Pergamon Press, London, in press.

4. KEILIN, D., AND MANN, T., Biochem.J.,34, 1163 (1940). 5. WAYGOOD, E.R.,in S.P. COLOWICKAND N.O.KAPLAN (Edi-

tors), Methods in enzymology, Vol. II, Academic Press, Inc., New York, 1955, p. 836.

6. LINDSKOG, S., Biochim. et Biophys. Acta, 39, 218 (1960). 7. DAVIS.R.P..~~ P.D. BOYER. H. LARDY. AND K. MYRBBCK

(Editors), !bhe enzymes, VoZ. e, Ed. 2, Academic Press, Inc., New York, 1961, p. 545.

8. SCOTT, D. A., AND MENDIVE, J. R., J. Biol. Chem., 140, 445 (1941).

9. TUPPER, R., WATTS, R. W. E., AND WORMALL, A., Biochem. J., 50, 429 (1951).

10. VALLEE,B.L.,RUPLEY, J.A., COOMBS, T.L., AND NEURATH, H., J. Biol. Chem., 236, 64 (1960).

11. COLEMAN, J. E., AND VALLEE, B. L., J. Biol. Chem., 236, 390 (1960).

12. VALLEE, B. L., COOMBS, T. L., AND HOCH, F. L., J. Biol. Chem.‘, 236, PC45 (1969).

13. VALLEE. B. L.. WILLIAMS. R. J. P.. AND COLEMAN. J. E.. Naturk, 190, 633 (1961). '

14. MALMSTR~~M, B. G., Arch. Biochem. Biophys., 46, 345 (1953).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

April 1962 S. Lindskog and B. G. Malmstrdm 1137

15.

16.

17. 18. 19. 20.

21.

22.

23.

24.

25.

26. 27.

28. 29.

30.

MALMSTR~M, B. G., in D. GLICK (Editor), Methods of biochemi- cal analysis, Vol. III, Interscience Publishers, Inc., New York, 1956, p. 327.

SANDELL, E. B., Calorimetric determinations of traces of metals, Ed. 2, Interscience Publishers, Inc., New York, 1950, pp. 623; 300; 274.

BOYER, P. D., J. Am. Chem. Sot., 76, 4331 (1954). BEERS, R. F., JR., Biochem. J., 62, 492 (1956). DAVIS, R. P., J. Am. Chem. Sot., 81, 5674 (1959). KELLER, H., AND PETERS, U. H., 2. physiol. Chem., 317, 228

(1959). Ho, C., AND STURTEVANT, J. M., Biochem. Biophys. Research

Communs., 3, 20 (1960). SCHACHMAN, H. K., in S. P. COLOWICK AND N. 0. KAPLAN

(Editors), Methods in enzymology, VoZ. IV, Academic Press, Inc., New York, 1957, p. 32.

MALMSTR~M, B. G., AND VHNN~RD, T., J. Molecular Biol., 2, 118 (1960).

LOWRY, T. M., Optical rotatory power, Longmans, Green & Co., Ltd., London, 1935.

KOLTHOFF, I. M., LEUSSING, D. L., AND LEE, T. S., J. Am. Chem. Sot., 73, 390 (1951).

GUSTAFSSON, L., Talanta, 4, 236 (1960). VALLEE, B. L., in P. D. BOYER, H. LARDY, AND K. MYRB~CK

(Editors), The enzymes, VoZ. 111, Ed. 2, Academic Press, Inc., New York, 1960, p. 225.

VALLEE, B. L., Advances in Protein Chem., 10, 317 (1955). VALLEE, B. L., AND ALTSCHULE, M. D., Physiol. Revs., 29,

370 (1949). GURD, F. R. N., AND GOODMAN, D. S., J. Am. Chem. Sot., 14,

670 (1952).

31. MALMSTR~M, B. G., The mechanism of metal-ion activation of enzymes. Studies on enolase, Almqvist & Wiksells Interna- tional Booksellers, Uppsala, Sweden, 1956.

32. WILLIAMS, R. J. P., in P. D. BOYER, H. LARDY, AND K. MYR- BXCK (Editors), The enzymes, Vol. I, Ed. 2, Academic Press, Inc., New York, 1959, pi 391:

33. KELLER. H.. 2. vhusiol. Chem.. 320.21 0960). 34. KELLER; H.‘, AN; GOTTTJALD, ‘P., i. physioi. Chem., 318, 201

(1960). 35. WARNER, R. C., Trans. N. Y. Acad. Sci., 16, 182 (1954). 36. GURD, F. R. N., AND WILCOX, P. E., Advances in Protein

Chem., 11, 311 (1956). 37. NYMAN, P. O., Biochim. et Biophys. Acta, 62, 1 (1961). 38. RICKLI, E. E., AND EDSALL, J. T., J. Biol. Chem., 237, PC 258

(1962). 39. WILLIAMS, R. J. P., in P. DESNUELLE AND A. E. BRAUNSTEIN

(Editors), Symposium IV of the jifth international congress of biochemistry, Moscow, 196’1, Pergamon Press, London, in press.

40. ROSENBERG, A., The role of metal ions in the catalytic action of peptidases. Studies on carnosinase, Almqvist & Wiksells International Booksellers, Uppsala, Sweden, 1960.

41. K&I, J. H. R., AND VALLEE, B. L., J. Biol. Chem., 236, 3188 (1960).

42. RUPLEY, J. A., AND NEURATH, H., J. Biol. Chem., 236, 609 (1960).

43. MALMSTR~M, B. G., AND ROSENBERG, A., Advances in Enzymol. 21, 131 (1959).

4.4. WILLIAMS, R. J. P., Nature, 188, 322 (1969). 45. KELLER, H., GOTTWALD, P., AND WENDLING, N., Biochem.

Biophys. Research Communs., 3, 24 (1960).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Sven Lindskog and Bo G. MalmströmMetal Binding and Catalytic Activity in Bovine Carbonic Anhydrase

1962, 237:1129-1137.J. Biol. Chem.

http://www.jbc.org/content/237/4/1129.citation

Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/237/4/1129.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/content/237/4/1129.citation

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;237/4/1129&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/237/4/1129.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=237/4/1129&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/237/4/1129.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/237/4/1129.citation.full.html#ref-list-1

http://www.jbc.org/

Metal Binding and Catalytic Activity in Bovine Carbonic Anhydrase*

Documents

Transcript of Metal Binding and Catalytic Activity in Bovine Carbonic Anhydrase*