Evaluation of the extent of the binding site in human tissue kallikrein by synthetic substrates with sequences of human kininogen fragments

Abstract

We have synthesized internally quenched peptides spanning the Met379-Lys380 or Arg389-Ser390 bonds of human kininogen (hkng) that flank lysyl-bradykinin and have studied the kinetics of their hydrolysis by human tissue kallikrein. The kinetic data for the hydrolysis of the Met-Lys bond in substrates with an N-terminal extension showed that interactions up to position residue P10 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 extensions at sites P′4 to P′11. A similar pattern was observed for the cleavage of substrates containing an Arg-Ser bond because substrates extended up to residue P6 were hydrolysed with the highest kcat/Km values 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 Leu373 to Ile393 human kininogen fragment containing omicron-aminobenzoic acid (Abz) at the N-terminus and an amidated C-terminal carboxyl group Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg- Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Ile-NH2 (Abz-[Leu373-Ile393]-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 kcat/Km values at pH 9-9.5 for both substrates. The pH-dependent variations in this kinetic parameter were almost exclusively due to variations in kcat. A significant decrease in kcat/Km values was observed for the hydrolysis of Arg-Ser and Met-Lys bonds in the presence of 0.1 M NaCl. Because this effect was closely related to an increase in Km, it is likely that sodium competes with the positive charges of the substrate side chains for the same enzyme subsites.