Catalytic properties of serine proteases. 2. Comparison between human urinary kallikrein and human urokinase, bovine β-trypsin, bovine thrombin, and bovine α-chymotrypsin

Paolo Ascenzi, Enea Menegatti, Mario Guarneri, Fabrizio Bortolotti, Eraldo Antonini

Research output: Contribution to journalArticle

Abstract

The catalytic properties of several serine proteases acting on cationic substrates (bovine β-trypsin, bovine thrombin, human urinary kallikrein, and human urokinase) and noncationic substrates (bovine α-chymotrypsin) have been compared in steady-state and pre-steady-state experiments by using ester and anilide synthetic substrates. Arginine and lysine derivatives are equally good substrates for b. β-trypsin; b. thrombin and h.u. kallikrein prefer substrates containing arginine side chains; h. urokinase prefers substrates containing lysine. The preference of the various enzymes for the guanidinium or ammonium group is reflected by the different promoter effect that acetamidine or ethylamine has on the catalyzed hydrolysis of neutral substrates. Pre-steady-state data, analyzed in the framework of the three-step model, show that for b. β-trypsin, b. thrombin, h.u. kallikrein, and h. urokinase the acylation step (k2) is rate limiting above pH 6 and the deacylation step (k3) below pH 4 in the hydrolysis of ZLysONp and of ZAlaONp in the presence of acetamidine or ethylamine. In the catalyzed hydrolysis of ZAlaONp, in the absence of acetamidine or ethylamine, the acylation step (k2) is rate limiting all over the pH range from 3 to 8. The change in the rate-limiting step with pH is always absent, for the same substrates, in the b. α-chymotrypsin catalysis. The results of kinetic and spectral measurements indicate that b. β-trypsin, b. thrombin, h.u. kallikrein, and h. urokinase, but not b. α-chymotrypsin, contain a similarly located ionizable group with a pKa of 4.50 ± 0.1, in the free enzyme, the ionization of which affects the binding of cationic substrates and ligands, the spectral properties of the proteases, and the rate of the acylation step in catalysis.

Original languageEnglish
Pages (from-to)2483-2490
Number of pages8
JournalBiochemistry
Volume21
Issue number10
Publication statusPublished - 1982

ASJC Scopus subject areas

  • Biochemistry

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