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|Title:||EVALUATION OF THE EXTENT OF THE BINDING-SITE IN HUMAN TISSUE KALLIKREIN BY SYNTHETIC SUBSTRATES WITH SEQUENCES OF HUMAN KININOGEN FRAGMENTS|
|Authors:||Del Nery, Elaine [UNIFESP]|
Chagas, Jair Ribeiro [UNIFESP]
Juliano, Maria Aparecida [UNIFESP]
Prado, Eline Sant'Anna [UNIFESP]
Juliano, Luiz [UNIFESP]
Universidade Federal de São Paulo (UNIFESP)
|Citation:||Biochemical Journal. London: Portland Press, v. 312, n. 1, p. 233-238, 1995.|
|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.|
|Appears in Collections:||Artigo|
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