EFFECTS OF PROTONS ON THE THROMBIN-FIBRINOGEN INTERACTION

Amidase activity of human a-thrombin toward the synthetic substrate To syl-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-pe rturbation method allowed us to compute the equilibrium binding consta nt along with the rate constants for the acylation and deacylation rea ctions. 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 substr ate is controlled by two ionizable groups having pK(a) values of 7.5 a nd 8.7 in the free enzyme and 6.3 and 9.8 in the Michaelis adduct. The se two groups were found to control the acylation process as well. Thr ombin-fibrinogen interaction has been studied by measurements of stead y-state hydrolysis of the synthetic substrate Phe-pipecolyl-Arg-NH-Ph in the presence of fibrinogen, used as a competitive inhibitor. This m ethod allowed us to measure the K-m of thrombin-fibrinogen interaction . The values of K-m computed at different solution viscosities were us ed in order to calculate the equilibrium dissociation constant and bot h k(2)/k(3) and k(2)/k(-1) ratios. The same residues that were found t o control binding of Tosyl-Gly-Pro-Arg-NH-Ph to alpha-thrombin, do mod ulate binding of fibrinogen as well. These residues shift their pK(a) 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 k(cat)/K-m as a f unction of pH has been obtained by HPLC measurements of fibrinopeptide s release. The k(cat)/K-m values along with the ratio k(2)/k(-1), deri ved from viscometric experiments, allowed us to calculate the forward- rate constant, k(+1), for the thrombin-fibrinogen interaction. The ass ociation process was found to depend on pH, namely in the alkaline reg ion. The results for Tosyl-Gly-Pro-Arg-NH-Ph and fibrinogen are compar ed and discussed on the basis of the structural elements which differe ntiate the interactions of these substrates with human alpha-thrombin.