MODULATION OF HUMAN ALPHA-THROMBIN ACTIVITY WITH PHOSPHONATE ESTER INHIBITORS

Enantiomers of 4-nitrophenyl 4-X-phenacyl methylphosphonate eaters (X = H, PMN; CH3; and CH3O) inactivate human alpha-thrombin with rate con stants 4-235 M-1 s(-1) in pH 6.5, 0.025 M citrate buffer, and 0.15 M N aCl at 7.0+/-0.1 degrees C. Stereoselectivity of the inactivation of t hrombin is 2-39 and favors the levorotatory enantiomers. The pH-depend ence of inactivation of thrombin by (-)-PMN is sigmoidal and consisten t with the participation of a catalytic residue with a pK(a) of 8.0+/- 0.1 in 0.15 M NaCl and a pK(a) of 7.4+/-0.2 in 0.15 M choline chloride in the nucleophilic attack of the catalytic Ser at phosphorus. The so lvent isotope effect on k(i)/K-i in the pH-independent region of the r eaction 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 intr amolecular reaction. The pH dependence of reactivation is significant from the adduct formed with (-)-CH3O-PMN and (-)-CH3-PMN and less so f rom the adducts formed with the other enantiomers of the inhibitors. K inetic pKs similar to 7.2, with the exception of the adducts with (-)- PMN and (-)-CH3O-PMN, indicate that a pH-dependent conformational chan ge affects the rate of dephosphonylation. A structural interpretation of the stereoselectivity and other mechanistic features is provided ba sed on the energy-optimized structures of the adducts. Pharmaco-medica l use of human alpha-thrombin covalently modified by the PMNs is sugge sted. (C) 1997 Elsevier Science Ltd.