The linkage between binding of the C-terminal domain of hirudin and amidase activity in human α-thrombin

Abstract

A method derived from the analysis of viscosity effects on the hydrolysis of the amide substrates D-phenylalanylpipecolylarginine-p-nitroaniline, tosylglycylprolylarginine-p-nitroanaline and cyclohexylglycylalanylarginine-p-nitroalanine by human α-thrombin was developed to dissect the Michaelis-Menten parameters K(m) and K(cat) 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) (K(d) ~ 0.7 μM). In addition this peptide was found to competitively inhibit thrombin-fibrinogen interaction with a K(i) 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.