Catalytic properties of human urinary kallikrein. 1

Publisher Website
Google Scholar
Abstract
Kinetic studies were carried out with a well-characterized preparation of human urinary (h.u.) kallikrein using chromogenic substrates. Steady-state and presteady-state data for h.u. kallikrein catalyzed hydrolysis of N.alpha.-carbobenzoxyl-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 were obtained under various conditions and were analyzed in the framework of the minimum 3-step mechanism. The pH dependencies of the kinetic parameters for the hydrolysis of ZLysONp and of ZAlaONp in the presence of saturating levels of ethylamine and acetamidine show that at acid pH values (.ltoreq. 4) the k3 step (deacylation) is rate limiting in catalysis; for pH values > 6, k2 (acylation) becomes rate limiting. 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 .apprx. 5-fold with pH between pH 5-3. The pH dependence of the spectral properties of free h.u. kallikrein reflects the ionization of a group with a pKa value of 4.45 .+-. 0.1. Apparently, similarly to bovine .beta.-trypsin, h.u. kallikrein catalysis involves an ionizable group which has a pKa .apprx. 4.5 in the free enzyme and a pKa .apprx. 3.7 in the enzyme bound to cationic substrates or ligands.