CATALYTIC PROPERTIES OF HUMAN LYS77-PLASMIN - A COMPARATIVE STEADY-STATE AND PRE-STEADY-STATE STUDY

Values of kinetic parameters of the hydrolysis of esters and p-nitroanilides of L-lysine and L-arginine catalyzed by the Lys77 form of human plasmin (EC 3.4.21.7) have been determined between pH 5.5 and 8 (I = 0.1 M) at 21 .+-. 0.5.degree. C. Over the whole pH range explored, Lys77-plasmin catalysis conforms to simple Michaelis-Menten kinetics, and steady-state and pre-steady-state data may be consistently fitted to the minimum three-step mechanism: E + S .dblarw. (k+1/k-1)E .cntdot. S .fwdarw. (k+2)E .cntdot. P + P1 .fwdarw. (k+3)E + P2. In spite of the higher specificity of lysyl derivatives of Lys77-plasmin rather than the arginyl ones, kinetic parameters also depend on the nature of the N-.alpha. substituent and/or of the alcoholic or p-nitroanilidic moiety of the substrate. Among the esters and anilides considered, ZLysONp shows the most favourable kinetic parameters and may be the substrate of choice of Lys77-plasmin, in that it allows the determination of the enzyme concentration as low as 2 .times. 10-9 M (about 1 .times. 10-3 CU/ml), at the optimum pH value (approx. 8). Between pH 5.5 and 8, the pH profiles of .kappa.cat and of .kappa.cat/Km for the Lys77-plasmin-catalyzed hydrolysis of ZLysONp and ZArgONp reflect the ionization of a single group (probably His-602 involved in the active site) with pKa values ranging between 6.4 and 6.6; at variance, values of Km are pH-independent.