The linkage between binding of the C-terminal domain of hirudin and amidase activity in human alpha-thrombin.
A method derived from the analysis of viscosity effects on the hydrolysis of the amide substrates D-phenylalanylpipecolyl-arginine-p-nitroaniline, tosylglycylprolylarginine-p-nitroanaline and cyclohexylglycylalanylarginine-p-nitroalanine by human alpha-thrombin was developed to dissect the Michaelis-Menten parameters Km and kcat into the individual rate constants of the binding, acylation and deacylation reactions. This method was used to analyse the effect of the C-terminal hirudin (residues 54-65) [hir-(54-65)] domain on the binding and hydrolysis of the three substrates. The results showed that the C-terminal hir-(54-65) fragment affects only the acylation rate, which is increased approx. 1.2-fold for all the substrates. Analysis of the dependence of acylation rate constants on hirudin-fragment concentration, allowed the determination of the equilibrium binding constant of C-terminal hir-(54-65) (Kd approximately 0.7 microM). In addition this peptide was found to competitively inhibit thrombin-fibrinogen interaction with a Ki which is in excellent agreement with the equilibrium constant derived from viscosity experiments. These results demonstrate that binding of hir-(54-65) to the fibrinogen recognition site of thrombin does not affect the equilibrium binding of amide substrates, but induces only a small increase in the acylation rate of the hydrolysis reaction.
de Cristofaro
R
R
Centro Ricerche Fisiopatologia dell'Emostasi, Università Cattolica S. Cuore, Roma, Italy.
Rocca
B
B
Bizzi
B
B
Landolfi
R
R
D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D005340:Fibrinogen / Q000378:metabolism; D006629:Hirudins / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D011485:Protein Binding; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D014783:Viscosity
D006629:Hirudins; D009842:Oligopeptides; D010446:Peptide Fragments; D005340:Fibrinogen; D013917:Thrombin; D000581:Amidohydrolases