The structure and function of carboxypeptidase CN.

Abstract

Carboxypeptidase CN from Citrus natsudaidai HAYATA is an acidic protease (M, 93000). The enzyme was inactivated by the incorporation of 1.0 mol of 32P-labeled diisopropylphosphofluoridate, 1.3 mol of 203HgCl2, or 2.2 mol of 110mAgNO3 per mol of enzyme at pH 5.5. Four or five radioactive peptides were isolated from a partial acid hydrolysate of the 32P-labeled enzyme. These peptides provided evidence indicating the amino acid sequence Glx-Gly-Asx-Ser-Gly-Gly-Glu-Leu-Val around the reactive serine residue. The enzyme reacted with 0.4 mol of 203Hg-labeled p-chloromercuribenzoate without appreciable loss of enzymatic activity. On the other hand, 1 mol of 203Hg-labeled p-chloromercuribenzoate was incorporated into the enzyme which had been denatured with 6 M guanidine hydrochloride and 8 M urea at pH 7.0 in the absence of dithiothreitol. Thus, the enzyme has one sulfhydryl group which possesses only poor reactivity. Photooxidation using methylene blue as a sensitizer caused a loss of enzymatic activity and specific destruction of approximately 1 mol of histidine residue per mol of enzyme. The photooxidized, p-bromophenacyl bromide-treated and HgCl2-treated enzyme failed to react with 32P-labeled diisopropylphosphofluoridate. From these findings, we inferred that one serine, one histidine, and the carboxyl groups are essential for catalytic activity.