Clostridium histolyticum collagenase: development of new thio ester, fluorogenic, and depsipeptide substrates and new inhibitors.
A new series of thio ester, depsipeptide, and peptide substrates have been synthesized for the bacterial enzyme Clostridium histolyticum collagenase. The hydrolysis of the depsipeptide substrate was followed on a pH stat, and thio ester hydrolysis was measured by inclusion of the chromogenic thiol reagent 4,4'-dithiopyridine in the assay mixture. The best thio ester substrate, Boc-Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba, had a kcat/KM of 63 000 M-1 s-1, while several shorter thio ester sequences were inactive as substrates. In general, the peptide analogues of all the reactive thio ester substrates were shown to be hydrolyzed 5-10 times faster by collagenase. In one 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 two 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 upon 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 reversible inhibitors were developed by the attachment of zinc 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 K1 (0.02 mM).(ABSTRACT TRUNCATED AT 250 WORDS)
Vencill
C F
CF
Rasnick
D
D
Crumley
K V
KV
Nishino
N
N
Powers
J C
JC
D003013:Clostridium / Q000201:enzymology*; D004952:Esters; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds
D004952:Esters; D013438:Sulfhydryl Compounds; D003012:Microbial Collagenase