Effects of protons on the thrombin‐fibrinogen interaction

Abstract

Amidase activity of human alpha-thrombin toward the synthetic substrate Tosyl-Gly-Pro-Arg-NH-Ph and fibrinogen has been studied as a function of pH at t = 25 degrees C, under steady-state conditions. A viscosity-perturbation method allowed us to compute the equilibrium binding constant along with the rate constants for the acylation and deacylation reactions. The ionization constants for the groups affecting binding and hydrolysis of the synthetic substrate were measured by application of linkage thermodynamics principles. The binding of the synthetic substrate is controlled by two ionizable groups having pKa values of 7.5 and 8.7 in the free enzyme and 6.3 and 9.8 in the Michaelis adduct. These two groups were found to control the acylation process as well. Thrombin-fibrinogen interaction has been studied by measurements of steady-state hydrolysis of the synthetic substrate Phe-pipecolyl-Arg-NH-Ph in the presence of fibrinogen, used as a competitive inhibitor. This method allowed us to measure the Km of thrombin-fibrinogen interaction. The values of Km computed at different solution viscosities were used in order to calculate the equilibrium dissociation constant and both k2/k3 and k2/k-1 ratios. The same residues that were found to control binding of Tosyl-Gly-Pro-Arg-NH-Ph to alpha-thrombin, do modulate binding of fibrinogen as well. These residues shift their pKa values upon the formation of the Michaelis adduct from 7.5 to 5.7 and from 8.7 to 9.7, respectively. Furthermore the ratio kcat/Km as a function of pH has been obtained by HPLC measurements of fibrinopeptides release. The kcat/Km values along with the ratio k2/k-1, derived from viscometric experiments, allowed us to calculate the forward-rate constant, k+1, for the thrombin-fibrinogen interaction. The association process was found to depend on pH, namely in the alkaline region. The results for Tosyl-Gly-Pro-Arg-NH-Ph and fibrinogen are compared and discussed on the basis of the structural elements which differentiate the interactions of these substrates with human alpha-thrombin.