Action of serine carboxypeptidases on endopeptidase substrates, peptide‐4‐methyl‐coumaryl‐7‐amides

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Abstract
Carboxypeptidase Y hydrolyzed N‐substituted peptide‐4‐methylcoumarin‐7‐amides (peptide‐NH‐Mec) at pH 7 by releasing 7‐amino‐4‐methylcoumarin (NH2‐Mec) which was then followed by carboxypeptidase action. In particular, a chymotrypsin‐directed substrate, Suc‐Leu‐Leu‐Val‐Tyr‐NH‐Mec, was hydrolyzed by the enzyme with a second‐order rate constant of 7200 M−1 S−1, which is compatible with the rate for an anilide substrate and some N‐substituted dipeptides. The activity was completely inhibited by phenylmethylsulfonyl fluoride and competitively depressed by the presence of an N‐substituted dipeptide. Dependences of kinetic parameters on pH were different from those of carboxypeptidase, esterase, amidase, and anilidase activities. Carboxypeptidases P from Penicillium janthinellum and W from wheat also hydrolyzed some of these peptide‐NH‐Mec derivatives in a similar manner but at a rather low rate. Thus, the NH2‐Mec‐releasing activity may be considered to be intrinsic to serine carboxypeptidases in general. Taking into consideration this endopeptidase‐like activity of serine carboxypeptidases, fluorogenic substrates should be used carefully to specify endopeptidases in crude extracts.