Active site mapping of the serine proteases human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II. Bovine chymotrypsin A alpha, and Staphylococcus aureus protease V-8 using tripeptide thiobenzyl ester substrates.
The primary subsite specificities of human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II, bovine chymotrypsin A alpha, and the protease from strain V-8 of Staphylococcus aureus have been mapped with a series of tripeptide thiobenzyl ester substrates of the general formula Boc-Ala-Ala-AA-SBzl, where AA represents one of 13 amino acids. In addition, the effects of a P2 Pro and P4 methoxysuccinyl and succinyl groups were investigated. In an attempt to introduce specificity and/or reactivity into the substrate Boc-Ala-Ala-Leu-SBzl(X), the 4-chloro-, 4-nitro-, and 4-methoxythiobenzyl ester derivatives were studied. Enzymatic hydrolyses of the substrates were measured in the presence of 4,4'-dithiobis(pyridine) or 5,5'-dithiobis(2-nitrobenzoic acid), which provided a highly sensitive assay method for free thiol. The thio esters were excellent substrates for the enzymes tested, and in many cases, the best substrates reported here have kcat/KM values higher than those reported previously. The best substrate for human leukocyte elastase was Boc-Ala-Pro-Nva-SBzl(Cl), which has a kcat/KM of 130 X 10(6) M-1 s-1. A very reactive rat mast cell protease substrate, Boc-Ala-Ala-Leu-SBzl(NO2), was also found. The S. aureus V-8 protease was the most specific enzyme tested since it hydrolyzed only Boc-Ala-Ala-Glu-SBzl. Substituents on the thiobenzyl ester moiety of Boc-Ala-Ala-Leu-SBzl resulted in decreased KM values with human leukocyte elastase and rat mast cell protease I when compared to the unsubstituted derivative.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001665:Binding Sites; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D008407:Mast Cells / Q000201:enzymology*; D008666:Metalloendopeptidases; D009842:Oligopeptides / Q000138:chemical synthesis*; D010179:Pancreas / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000378:metabolism; D051381:Rats; D012697:Serine Endopeptidases; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity
D007202:Indicators and Reagents; D009842:Oligopeptides; D002403:Cathepsins; D010450:Endopeptidases; C025097:Escherichia coli periplasmic proteinase; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; C534094:CTSG protein, human; D056649:Cathepsin G; C534097:Ctsg protein, rat; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus