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Enzymatically inactive, exchange-inert Co(III)-carboxypeptidase A: role of inner sphere coordination in peptide and
Abstract:
Catalytically inactive, exchange-inert Co(III)-carboxypeptidase A has been prepared by reaction of Co(II)-carboxypeptidase A with the active-site-directed oxidizing agent m-chloroperbenzoic acid. Co(III)-carboxypeptidase A, isolated by affinity gel filtration chromatography, has the same amino acid composition and molecular weight as the starting material and contains 0.95 g-atom/mol of cobalt and 0.01 g-atom/mol of zinc. Its electron paramagnetic resonance, circular dichroic, magnetic circular dichroic, and visible absorption spectra are consistent with those of octahedral Co(III) model complexes. Co(III)-caboxypeptidase A is essentially devoid of catalytic activity toward both peptide and ester substrates of the native enzyme, and stopped-flow fluorescence studies with dansylated substrates show that it binds peptides, but not esters. Furthermore, the protein does not react with either type of substrate to yield a single turnover. The implications of these findings to the mechanism of action of carboxypeptidase A are discussed in the light of the "metal-carbonyl" and "metal-hydroxide" hypotheses. Since Co(III)-carboxypeptidase A does not bind esters, inner-sphere coordination to the metal appears to be necessary for ester binding. All attempts to prepare Co(III)-carboxypeptidase A by treatment of Co(II)-carboxypeptidase A with hydrogen peroxide according to previously published procedures (Kang, E.P., Storm, C.B., & Carson, F.W. (1975) J. Am. Chem. Soc. 97, 6723) have been unsuccessful, and the present results do not confirm earlier reports that Co(III)-carboxypeptidase A exhibits esterase activity or that its activity is dependent on the method of preparation of the precursor Co(II)-carboxypeptidase A (Jones, M.M., Hunt, J.B., Storm, C.B., Evans, P.S., Carson, F.W. & Pauli, W.J. (1977) Biochem. Biophys. Res. Commun. 75, 253). These findings call for a reexamination of mechanistic conclusions based on the assumption that Co(III)-carboxypeptidase A is an active esterase.