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

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Abstract
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 S. aureus were 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 [and Boc = tert-butyloxycarbonyl]. 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) [Nva = norvaline], which has a kcat/Km of 130 .times. 106 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. The 4-nitro group caused a 7-fold increase in reactivity (kcat/Km) toward rat mast cell protease I, and the 4-chloro derivative was 3.3-fold more reactive than the unsubstituted derivative toward human leukocyte elastase. This series could prove useful in mapping the active site of newly isolated serine proteases and has the advantage of being highly sensitive, thereby requiring smaller quantities of enzyme than other methods.
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