Enzymatically inactive, exchange-inert cobalt(III)-carboxypeptidase A: role of inner sphere coordination in peptide and ester catalysis

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.

A retroviral-like metal binding motif in an aminoacyl-tRNA synthetase is important for tRNA recognition.

“…In these experiments, the oxidation of Co2+ was confirmed by loss of the characteristic electron paramagnetic resonance signal at g = 4.6 (data not shown). The remaining aminoacylation activity of the oxidized cobalt-containing enzyme may be due to incomplete substitution of alanine tRNA synthetase with cobalt and, additionally or alternatively, to incomplete conversion to the Co3`form (28,29). Because the native enzyme's activity is not substantially affected by treatment with H202 and because the adenylate synthesis activity of the Co2+-substituted enzyme is not altered by the oxidation, the loss of aminoacylation activity by the Co3+-enzyme is probably not due to a global effect of oxidation on enzyme conformation.…”

A metal‐binding motif implicated in RNA recognition by an aminoacyl‐tRNA synthetase and by a retroviral gene product

“…The change in metal ion co-ordination may disturb a specific metal-RNA interaction or alter the configuration of the motif that interacts with the acceptor helix; because Co(lll) is inert to ligand exchange within the first co-ordination sphere (Van Wart and Vallee, 1978;Van Wart, 1988}, these results may Indicate that inner sphere co-ordination to the metal takes place during the second stage of catalysis (transfer of alanine to the 3' end '^'…”

“…The enzyme-cobalt co-ordination in the metal-binding site can be altered by oxidation of Co(ll) to Co(lll) in situ by treatment with hydrogen peroxide (Van Wart and Valtee, 1978;Van Wart, 1988), The aminoacylation activity of the Co(ll) enzyme is inhibited dramatically upon oxidation to Co(lll), but the ability of the enzyme to catalyse formation of the alanyl-adenylate intermediate is unaffected (Miller and Schimmel, 1992). The change in metal ion co-ordination may disturb a specific metal-RNA interaction or alter the configuration of the motif that interacts with the acceptor helix; because Co(lll) is inert to ligand exchange within the first co-ordination sphere (Van Wart and Vallee, 1978;Van Wart, 1988}, these results may Indicate that inner sphere co-ordination to the metal takes place during the second stage of catalysis (transfer of alanine to the 3' end '^'…”

A comparison of Zn(II) and Co(II) in the kinetics of inactivation of aminoacylase by 1,10-phenanthroline and reconstitution of the apoenzyme

“…As the d-shell electrons are filled for Zn(II), it is completely without absorption, e.s.r. or other signals for spectroscopic studies of the role of the metal ion in the catalytic process; paramagnetic metals have been used as probes for such studies (Latt and Vallee, 1971;Van Wart and Vallee, 1978;Vallee and Galdes, 1984;Kuo and Makinen, 1985;Heese et al, 1990). Co(II) is sufficiently similar to Zn so that it can replace Zn in most of the Zn enzymes with nearly full catalytic activity, and consequently it has been extensively used as a spectroscopic probe for studies on the structure of the active sites of Zn enzymes and the role of the metal ion during catalysis (Vallee and Wacker, 1970;Vallee and Holmquist, 1980;Geoghegan et al, 1983).…”