Aug 8, 1978

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

Biochemistry
H E Van Wart, B L Vallee

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 bi...Continue Reading

Mentioned in this Paper

Carboxypeptidase A
Hydrogen Peroxide
Apoenzymes
Sodium Dithionite
Complex (molecular entity)
Carboxypeptidase A Activity
Fluorescence Spectroscopy
Gel Chromatography
Enzyme Activity
Protein Conformation

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