Clostridium histolyticum collagenase: development of new thio ester, fluorogenic, and depsipeptide substrates and new inhibitors

Abstract

A new series of thio ester, depsipeptide and peptide substrates were synthesized for the bacterial enzyme C. histolyticum collagenase. The hydrolysis of the depsipeptide substrate was followed on a pH stat, and thioester hydrolysis was measured by inclusion of the chromogenic thiol reagent 4,4''-dithiopyridine in the assay mixture. The best thioester substrate, Boc-Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba, had a kcat/KM of 63,000 M-1 s-1, while several shorter thioester sequences were inactive as substrates. In general, the peptide analogs of all the reactive thioester substrates were shown to be hydrolyzed 5-10 times faster by collagenase. In 1 case (Z-Gly-Pro-Leu-Gly-Pro-NH2) where a comparison was made, the peptide substrate was, respectively, 8- and 106-fold more readily hydrolyzed than the corresponding thio ester and ester substrates. Cleavages of the 2 fluorescence-quench substrates Abz-Gly-Pro-Leu-Gly-Pro-Nba and Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba could be easily followed fluorogenically since a 5-10-fold increase in fluorescence occurred on hydrolysis. The fluorescent peptide substrate is the best synthetic substrate known for C. histolyticum collagenase with a Kcat/KM value of 490,000 M-1 s-1. A series of new reservsible inhibitors were developed by the attachment of Zn ligating groups (hydroxamic acid, carboxymethyl and thiol) to various peptide sequences specific for C. histolyticum collagenase. The shorter peptides designed to bind to either the P3-P1 or P1''-P3'' subsites were poor to moderate inhibitors. The thiol HSCH2CH2CO-Pro-Nba had the lowest Ki (0.02 mM). Elongation of N-hydroxy peptide sequences to interact with the P3-P3'' subsites of the enzyme failed to yield better inhibitors. None of the potential irreversible inhibitor structures, which contained ClCH2CO.sbd. or CH2.dbd.CH.sbd.CO.sbd. groups attached to peptides, proved to be effective.