The pH dependence of pre-steady-state and steady-state kinetics for the porcine pancreatic beta-kallikrein-B-catalyzed hydrolysis of N-alpha-carbobenzoxy-L-arginine p-nitrophenyl ester.
Pre-steady-state and steady-state kinetics of the porcine pancreatic beta-kallikrein-B (EC 3.4.21.35) catalyzed hydrolysis of ZArgONp have been determined between pH 2.4 and 8. The results are consistent with a minimum three-step mechanism involving an acyl-enzyme intermediate: (see formula). The formation of the E X S complex may be regarded as a pseudoequilibrium process; the minimum values for k+1 are 5.9 X 10(6) M-1 X s-1 (pH 5.5) and 9.4 X 10(5) M-1 X s-1 (pH 2.4) and that for k-1 is 600 s-1. The value of k-1/k+1 (= Ks) changes from 102 microM at pH greater than or equal to 5.5 to 638 microM at pH less than 2.4. The pH dependence of k+2 conforms to two ionizing groups, in the E X S complex, with pKa values of 3.4 +/- 0.1 and 7.05 +/- 0.10. The pH profile of k+2/Ks (= kcat/Km) reflects the ionization of two groups, in the free enzyme, with pKa values of 4.2 +/- 0.1 and 7.05 +/- 0.10. The pH dependence of k+3 implicates two ionizing groups in the deacylation step with pKa values of 4.6 +/- 0.1 and 7.0 +/- 0.1. At acid pH values (pH 2.4-4.4), k+3 is rate-limiting in catalysis, whereas for pH values higher than 4.4, k+2 becomes rate-limiting. The observed neutral and acid ionizations probably reflect the acid-base equilibrium of His-57 and Asp-189 involved in the central site of beta-kallikrein-B. The structural basis for the specificity and catalytic behaviour of this proteinase are discussed and a role for Ser-226 is pinpointed.
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007610:Kallikreins / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D010179:Pancreas / Q000201:enzymology*; D013552:Swine
D046911:Macromolecular Substances; C040761:N-benzyloxycarbonylarginine 4-nitrophenyl ester; D001120:Arginine; D007610:Kallikreins