Effects of pH and inhibitors on some properties related to metal binding in bovine carbonic anhydrase.

Carbonic anhydrase contains one firmly bound zinc ion per enzyme molecule (1). The metal ion, which can be dissociated in the presence of a chelating agent, is essential for catalytic activity, and it has been suggested that it is a part of the active site (2). Some other divalent metal ions also activate the metal-free enzyme. For example, Co2+ gives 45oj, of the activity of the native enzyme (2). Because of its weaker binding, Co2+ can easily be displaced from the apoenzyme by Zn2+; concomitantly the activity rises to the value for the native enzyme, while the total amount of bound metal remains close to one ion per molecule, suggesting a common chelating site for both metal ions (2). The aim of the work presented in this communication has been to obtain further information as to the mode of metal coordination in carbonic anhydrase and the role of the metal ion in the catalytic mechanism of the enzyme. The complex visible absorption spectrum of the intensely reddish blue Co2+-derivative has been used as a means of observing the immediate neighborhood of the metal ion as influenced by changes in pH and inhibitor concentration. The results indicate that one of the ligand groups, although linked to the metal ion at basic pH, becomes uncoupled in neutral and weakly acid solution by “preferring” a proton. Evidence has been obtained that inhibitors of the “metal-poison” type as well as sulfonamides (cf. (3)) interact directly with the metal ion, but are displaced from it as the pH is increased. This has been interpreted as a competition for a coordination site of the metal ion between the inhibitor molecule and the same ligand group. In titration studies on the native enzyme some corresponding effects have been observed and the results are consistent with this mechanism.