Metal Binding and Catalytic Activity in Bovine Carbonic Anhydrase

Abstract

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 investigations 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 metalloenzyme, 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 metalbinding site have recently been published (12, 13). The results presented in this report show that in many respects carbonic anhydrase behaves similarly to carboxypeptidase.