Bjr. Forbes et Ga. Gordon, MECHANISM AND MECHANISM-BASED INACTIVATION OF 4-HYDROXYPHENYLPYRUVATEDIOXYGENASE, Bioorganic chemistry, 22(4), 1994, pp. 343-361
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.