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

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

Author Del Nery, Elaine Autor UNIFESP Google Scholar
Chagas, Jair Ribeiro Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Prado, Eline Sant'Anna Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract We have synthesized internally quenched peptides spanning the Met(379)-Lys(380) Or Arg(389)-Ser(390) 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 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 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 P-6 were hydrolysed with the highest k(cat)/K-m 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 Leu(373) to Ile(393) human kininogen fragment containing o-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-[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 parameter were almost exclusively due to variations in k(cat). A significant decrease in k(cat)/K-m 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 K-m, it is likely that sodium competes with the positive charges of the substrate side chains for the same enzyme subsites.
Language English
Date 1995-11-15
Published in Biochemical Journal. London: Portland Press, v. 312, n. 1, p. 233-238, 1995.
ISSN 0264-6021 (Sherpa/Romeo, impact factor)
Publisher Portland Press
Extent 233-238
Origin http://dx.doi.org/10.1042/bj3120233
Access rights Open access Open Access
Type Article
Web of Science ID WOS:A1995TF37100031
URI http://repositorio.unifesp.br/11600/42798

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