Interaction of carboxylate inhibitors with the active site of nickel(II) carboxypeptidase A

Abstract

The binding of several carboxylate inhibitors to nickel(II)-substituted carboxypeptidase A, NiCPA, has been investigated through 1H, 13C NMR and electronic absorption spectroscopies. Both β-phenylpropionate and phenylacetate interact with NiCPA forming two complexes of stoichiometries 1 : 1 and 1 : 2 and their affinity constants were determined. Whereas the first inhibitor molecule binds at a non-metallic site, the second binds directly to the metal ion in slow exchange on the chemical shift time-scale. Proton nuclear Overhauser effect measurements have been performed on the 1 : 2 β-phenylpropionate complex, allowing a full correlation between the isotropically shifted signals. From the 1H T1 values of the meta-like protons of the co-ordinated histidines and the molar absorbances of the 1 : 2 complexes formed, five-co-ordination for the nickel ion is suggested. The NMR data indicate that upon binding of the carboxylate to the metal ion a conformational change occurs. In contrast, acetate displays no evidence for more than a single binding mode to the nickel enzyme. Thus 1H and 13C NMR data indicate that acetate binds to the metal ion forming a 1 : 1 complex in a fast-exchange regime. The Ni ⋯13C distance, r= 2.7 Å, calculated by means of the Solomon equation, is consistent with direct co-ordination of the acetate to the metal ion.