Metal ion effects on target sites of modification in metallocarboxypeptidase B.
Native carboxypeptidase B and its Co2+-substituted derivative were oxidized by the active-site-directed agent m-chloroperbenzoic acid. The following results were obtained a) In the cobalt enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Peptide or ester pseudo-substrates protected the cobalt enzyme but not the zinc enzyme against inactivation. b) Upon oxidation and formation of Co3+, cleavage of peptide bonds occurred in the cobalt enzyme but not in the zinc enzyme. Both enzymes retained their original metal content. c) Following oxidation of the enzymes, amino acid analysis revealed a modification of a methionyl residue in the zinc enzyme only; the cobalt enzyme, on the other hand, showed a modification of a histidyl residue. d) Peptide mapping of the enzymes after cleavage by cyanogen bromide indicated that two methionyl peptides were missing in the oxidized zinc enzyme. These peptides point to Met-64 as the site of modification. The peptide map of the oxidized cobalt enzyme was similar to that of the unmodified native (i.e., zinc) enzyme. These studies indicate that the specific metal ion present in the enzyme imposes certain structural and functional differences on the active site, leading to differing reactivities of specific amino acid residues and to a different alignment of the active-site-directed reagent in the two enzymes.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism*; D002723:Chlorobenzoates / Q000494:pharmacology; D003035:Cobalt / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D013552:Swine; D015032:Zinc / Q000378:metabolism*
D000596:Amino Acids; D002723:Chlorobenzoates; D010446:Peptide Fragments; D003035:Cobalt; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; C000433:3-chloroperbenzoic acid; D015032:Zinc