Catalytic properties of human urinary kallikrein.
Kinetic studies have been carried out with a well-characterized preparation of human urinary (h.u.) kallikrein using chromogenic substrates. Steady-state and pre-steady-state data for h.u. kallikrein catalyzed hydrolysis of N alpha-carbobenzoxy-L-lysine p-nitrophenyl ester (ZLysONp) and of N alpha-carbobenzoxy-L-alanine p-nitrophenyl ester (ZAlaONp) in the presence and absence of ethylamine and acetamidine have been obtained under various conditions and have been analyzed in the framework of the minimum three-step mechanism: (formula see text) The pH dependencies of the kinetic parameters for the hydrolysis of ZLysONp and ZAlaONp in the presence of saturating levels of ethylamine and acetamidine show that at acid pH values (less than or equal to 4) the k3 step (deacylation) is rate limiting in catalysis, whereas for pH values greater than or equal to 6, k2 (acylation) becomes rate limiting. On the other hand, the acylation step is rate limiting in the enzymatic hydrolysis of ZAlaONp over the whole pH range explored. Saturating concentrations of acetamidine increase, more than those of ethylamine, kcat for the hydrolysis of ZAlaONp. The affinity of h.u. kallikrein for acetamidine and ethylamine changes about 5-fold with pH between pH 5 and 3. The pH dependence of the spectral properties of free hu. kallikrein reflects the ionization of a group with a pKa value of 4.45 +/- 0.1. The results point out that, similarly to bovine beta-trypsin, h.u. kallikrein catalysis involves an ionizable group which has a pKa of about 4.5 in the free enzyme and a pKa of about 3.7 in the enzyme bound to cationic substrates or ligands.
D000578:Amidines / Q000494:pharmacology; D002863:Chromogenic Compounds; D005021:Ethylamines / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007610:Kallikreins / Q000652:urine*; D007700:Kinetics; D008956:Models, Chemical
D000578:Amidines; D002863:Chromogenic Compounds; D005021:Ethylamines; D007610:Kallikreins; C023731:acetamidine