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

Abstract

The catalytic properties of several serine proteases acting on cationic substrates (bovine of beta-trypsin, bovine thrombin, human urinary kallikrein, and human urokinase) and noncationic substrates (bovine alpha-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. beta-trypsin; b. thrombin and h.u. kallikrein prefer substrates containing arginine side chains; h. urokinase prefers substrate containing lysine. The preference of the various enzymes for the guanidinium or ammonium group in reflected by the different promotor 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. beta-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. alpha-chymotrypsin catalysis. The results of kinetic and spectral measurements indicate that b. beta-trypsin, b. thrombin, h.u. kallikrein, and h. urokinase, but not b. alpha-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 pancreas, and the rate of the acylation step in catalysis.