MECHANISM AND MECHANISM-BASED INACTIVATION OF 4-HYDROXYPHENYLPYRUVATEDIOXYGENASE

Six substrate analogs of 4-hydroxyphenylpyruvate, specifically pentafl uorophenylpyruvate, ydroxytetrafluorophenylpyruvate,2-thienylpyruvate, 3-thienylpyruvate, thiophenol oxalate, and p-thiocresoloxalate were s ynthesized and their interactions with porcine liver 4-hydroxyphenylpy ruvate dioxygenase investigated. Both pentafluorophenylpyruvate and th iophenol oxalate are competitive inhibitors of the enzyme with Ki valu es of 14 and 150 mu M, respectively, but p-thiocresol oxalate has no e ffect on the enzymic activity. The other three substrate analogs are b oth substrates and mechanism-based inactivators of the enzyme with the following kinetic characteristics (compound, K-m, V-max, k(mact), K', partition ratio) at pH 6.0, 37 degrees C, and an air atmosphere: 4-hy droxytetrafluorophenylpyruvate, 50 mu M, 1.9 mkat/ kg, 1.5/min, 70 mu M 4.2; 2-thienylpyruvate, 500 mu M, 7.8 mkat/kg, 0.6/min, 400 CLM, 41; 3thienylpymvate, 250 mu M, 2 9 mkatikg, 0.6/min, 300 CLM, 22. When in activated, the dioxygenase was found to contain per mole of active enz yme, 0.78 mol of label from 3-thienyt3 [3H]pyruvate and 0.85 mol of la bel from 4-hydroxytetrafluorophenyl-3 [3H]pyruvate. The product formed from the enzyme-catalyzed oxidation of 3-thienylpyruvate was determin ed to be 3-carboxymethyl-3-thiolene-2-one. The implication of these re sults to the mechanism of the dioxygenase is considered, (C) 1994 Acad emic Press, Inc.