A new substrate and two inhibitors applicable for thermitase, subtilisin BPN' and alpha-chymotrypsin. Comparison of kinetic parameters with customary substrates and inhibitors.
A new chromogenic substrate and two inhibitors with the common peptide sequence X-Ala-Ala-Phe-Y have been synthesized, and were found to be of higher efficiency as hitherto available customary substrates and inhibitors for thermitase, subtilisin BPN' and alpha-chymotrypsin. The proteolytic coefficient kcat/Km for the hydrolysis of the substrate Suc-Ala-Ala-Phe-pNA is about 80 times higher in the case of thermitase and subtilisin BPN' and about 20 times higher for alpha-chymotrypsin compared with Suc-Ala3-pNA and Suc-Phe-pNA, respectively. The irreversible inhibitory effect of Z-Ala2-Phe-CH2Cl compared with Z-Phe-CH2Cl is 280 times greater for thermitase and subtilisin BPN' and 50 times for alpha-chymotrypsin. The corresponding methyl ketone Z-Ala2-Phe-CH3 is a high affinity competitive inhibitor for thermitase but not for the two other enzymes.
D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002863:Chromogenic Compounds / Q000378:metabolism / Q000494:pharmacology; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D002863:Chromogenic Compounds; D009842:Oligopeptides; D011480:Protease Inhibitors; C047113:benzyloxycarbonylalanyl-alanyl-phenylalanine-4-nitroanilide; C049105:succinyl-alanyl-alanyl-phenylalanine-4-nitroanilide; C047499:benzyloxycarbonylalanyl-alanyl phenylalanine chloromethyl ketone; D010450:Endopeptidases; D012697:Serine Endopeptidases; D013381:Subtilisins; C022728:thermitase; D002918:Chymotrypsin