Reversible modification of tissue-type plasminogen activator by methylphosphonate esters.
In spite of their rapid aqueous hydrolysis, 4-nitrophenyl 4-X-phenacyl methylphosphonates (X = H, (PMN) CH3, CH3O, Cl and NO2) inactivate many serine proteases of the pancreatic and blood coagulation systems efficiently. The rate constants, K/Ki, for the inactivation of tissue-type plasminogen activator enzyme (t-PA) are 470-750 M-1 S-1 with PMN, 4-CH3-PMN, and 4-CH3O-PMN in pH 7.8, 0.05 M Tris buffer at 7.0 +/- 0.5 degrees C, but t-PA cannot be inhibited with the 4-Cl and NO2 derivatives due to rapid competing hydrolysis. Enzyme activity returns from each enzyme-adduct at a characteristic rate, due to a self-catalyzed intramolecular reactivation process. The rate constants for spontaneous reactivation of t-PA from the adducts formed with the three inhibitors are K = 0.25-12.3 x 10(-2) min-1 at pH 7.4 and 25.0 +/- 0.1 degrees C and pH-dependent with an apparent pK approximately 8.3. The recovery of t-PA activity from the adducts in 40% human plasma buffered at pH 7.4 is the same or twice that in plain buffer. The presence of fibrin has a slight effect on inactivation but not on reactivation. The modulation of enzyme activity by reversible generation of the phosphonylated adducts has potential for medical application.
Zhao
O
O
Department of Chemistry, Catholic University of America, Washington, DC 20064, USA.
Kovach
I M
IM
D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000737:chemistry / Q000494:pharmacology*; D004952:Esters; D005343:Fibrinolytic Agents / Q000627:therapeutic use; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009203:Myocardial Infarction / Q000628:therapy; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000627:therapeutic use; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
D004793:Enzyme Reactivators; D004952:Esters; D005343:Fibrinolytic Agents; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C032627:methylphosphonic acid; D010959:Tissue Plasminogen Activator