Studies on zinc enzymes and models

Abstract

Carboxypeptidase A catalyzes the hydrolysis of peptides and related esters. For one special type of ester an anhydride intermediate has been suggested [1], but our work on 18O exchange reactions [2] indicated that normal peptide substrates do not use a mechanism involving an anhydride intermediate but instead proceed by direct hydrolysis. A loophole in this proof involves the ‘trapped water’ problem: 18O exchange with solvent might not be observed if the water molecule produced in one step is enzyme bound, and not equilibrated with solvent. Kinetic 18O isotope effect studies have now been performed to close this loophole and establish the true enzymatic mechanism. In kinetic isotope effect studies a reaction partitions between heavy and light water. Another partitioning of interest is between water and methanol. We find that even the unusual substrate which reportedly [1] forms an anhydride with carboxypeptidase A will not incorporate methanol. By contrast, model systems [3] for the zinc-catalyzed cleavage of an anhydride show a preference for methanolysis over hydrolysis. This contrasting behavior suggests that the enzymatic reaction involves two proton transfers. Carbonic anhydrase is a zinc enzyme catalyzing a deceptively simple hydration reaction. A variety of ligands have been prepared [4] which form zinc complexes related to the active site of the enzyme, but few of the complexes show striking catalytic ability. The special requirements for catalysis will be discussed.