EVALUATION OF THE EXTENT OF THE BINDING-SITE IN HUMAN TISSUE KALLIKREIN BY SYNTHETIC SUBSTRATES WITH SEQUENCES OF HUMAN KININOGEN FRAGMENTS

We have synthesized internally quenched peptides spanning the Met(379) -Lys(380) Or Arg(389)-Ser(390) bonds of human kininogen (hkng) that fl ank lysyl-bradykinin and have studied the kinetics of their hydrolysis by human tissue kallikrein. The kinetic data for the hydrolysis of th e Met-Lys bond in substrates with an N-terminal extension showed that interactions up to position residue P-10 contribute to the efficiency of cleavage. In contrast, there were no significant variations in the kinetic data for the hydrolysis of substrates with C-terminal extensio ns at sites P'(4) to P'(11). A similar pattern was observed for the cl eavage of substrates containing an Arg-Ser bond because substrates ext ended up to residue P-6 were hydrolysed with the highest k(cat)/K-m va lues in the series, whereas those extended to P'(11) on the C-terminal side had a lower susceptibility to hydrolysis. Time-course studies of hydrolysis by human and porcine tissue kallikreins of a Leu(373) to I le(393) human kininogen fragment containing o-aminobenzoic acid (Abz) at the N-terminus and an amidated C-terminal carboxyl group ro-Pro-Gly -Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Ile-NH2 (Abz-[Leu(373)-Ile(393)]-hkng -NH2) indicated that the cleavage of Met-Lys and Arg-Ser bonds in the same molecule occurs via the formation of independent enzyme-substrate complexes. The hydrolysis of Abz-F-R-S-S-R-Q-EDDnp [where EDDnp is N- (2,4-dinitrophenyl)ethylenediamine] and Abz-M-I-S-L-M-K-R-P-Q-EDDnp by human tissue kallikrein had maximal k(cat)/K-m values at pH 9-9.5 for both substrates. The pH-dependent variations in this kinetic paramete r were almost exclusively due to variations in k(cat). A significant d ecrease in k(cat)/K-m values was observed for the hydrolysis of Arg-Se r and Met-Lys bonds in the presence of 0.1 M NaCl. Because this effect was closely related to an increase in K-m, it is likely that sodium c ompetes with the positive charges of the substrate side chains for the same enzyme subsites.