Modulation of human alpha-thrombin activity with phosphonate ester inhibitors

Enantiomers of 4-nitrophenyl 4-X-phenacyl methylphosphonate esters (X = H, PMN; CH3; and CH3O) inactivate human alpha-thrombin with rate constants 4-235 M-1 s-1 in pH 6.5, 0.025 M citrate buffer, and 0.15 M NaCl at 7.0 +/- 0.1 degrees C. Stereoselectivity of the inactivation of thrombin is 2-39 and favors the levorotatory enantiomers. The pH-dependence of inactivation of thrombin by (-)-PMN is sigmoidal and consistent with the participation of a catalytic residue with a pKa of 8.0 +/- 0.1 in 0.15 M NaCl and a pKa of 7.4 +/- 0.2 in 0.15 M choline chloride in the nucleophilic attack of the catalytic Ser at phosphorus. The solvent isotope effect on ki/Ki in the pH-independent region of the reaction is 2.26 +/- 0.17. Thrombin activity returns from the adducts on the 2-7 h time scale at 25.0 +/- 0.1 degrees C via a self-catalyzed intramolecular reaction. The pH dependence of reactivation is significant from the adduct formed with (-)-CH3O-PMN and (-)-CH3-PMN and less so from the adducts formed with the other enantiomers of the inhibitors. Kinetic pKs approximately 7.2, with the exception of the adducts with (-)-PMN and (-)-CH3O-PMN, indicate that a pH-dependent conformational change affects the rate of dephosphonylation. A structural interpretation of the stereoselectivity and other mechanistic features is provided based on the energy-optimized structures of the adducts. Pharmaco-medical use of human alpha-thrombin covalently modified by the PMNs is suggested.