Mammalian Chymotrypsin-like Enzymes. Comparative Reactivities of Rat Mast Cell Proteases, Human and Dog Skin Chymases, and Human Cathepsin G with Peptide 4-Nitroanilide Substrates and with Peptide Chloromethyl Ketone and Sulfonyl Fluoride Inhibitors

James C. Powers, Takumi Tanaka, J. Wade Harper, Yoshihiro Minematsu, Larry Barker, Danforth Lincoln, Katherine V. Crumley, Jorma E. Fraki, Norman M. Schechter, Gerald G. Lazarus, Kiichiro Nakajima, Katsuhiro Nakashino, Hans Neurath, Richard G. Woodbury

Research output: Contribution to journalArticlepeer-review

Abstract

The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and α1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had K1 values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4S02F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.

Original languageEnglish (US)
Pages (from-to)2048-2058
Number of pages11
JournalBiochemistry
Volume24
Issue number8
DOIs
StatePublished - Apr 1 1985
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

Fingerprint

Dive into the research topics of 'Mammalian Chymotrypsin-like Enzymes. Comparative Reactivities of Rat Mast Cell Proteases, Human and Dog Skin Chymases, and Human Cathepsin G with Peptide 4-Nitroanilide Substrates and with Peptide Chloromethyl Ketone and Sulfonyl Fluoride Inhibitors'. Together they form a unique fingerprint.

Cite this