Action of serine carboxypeptidases on endopeptidase substrates, peptide-4-methyl-coumaryl-7-amides.
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.
D000596:Amino Acids / Q000032:analysis; D002268:Carboxypeptidases / Q000378:metabolism*; D043402:Cathepsin A; D003374:Coumarins / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D010407:Penicillium / Q000201:enzymology; D010455:Peptides / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology
D000596:Amino Acids; D003374:Coumarins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C025427:carboxypeptidase P; D002268:Carboxypeptidases; D010450:Endopeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase