Six substrate analogs of 4-hydroxyphenylpyruvate, specifically pentafluorophenylpyruvate, 4-hydroxytetrafluorophenylpyruvate, 2-thienylpyruvate, 3-thienylpyruvate, thiophenol oxalate, and (p)-thiocresol oxalate were synthesized and their interactions with porcine liver 4-hydroxyphenylpyruvate dioxygenase investigated. Both pentafluorophenylpyruvate and thiophenol oxalate are competitive inhibitors of the enzyme with (K_{1}) values of 14 and 150 (\mu \mathrm{M}), respectively, but (p)-thiocresol oxalate has no effect on the enzymic activity. The other three substrate analogs are both substrates and mechanism-based inactivators of the enzyme with the following kinetic characteristics (compound, (K_{m}, V_{\max }, k_{\text {inact }}, K^{\prime}), partition ratio) at (\mathrm{pH} 6.0,37^{\circ} \mathrm{C}), and an air atmosphere: 4-hydroxytetrafluorophenylpyruvate, (50 \mu \mathrm{M}, 1.9 \mathrm{mkat} /) kg. (1.5 / \mathrm{min}, 70 \mu \mathrm{M}, 4.2 ; 2)-thienylpyruvate, (500 \mu \mathrm{M}, 7.8 \mathrm{mkat} / \mathrm{kg}, 0.6 / \mathrm{min}, 400 \mu \mathrm{M}, 41 ; 3-) thienylpyruvate, (250 \mu \mathrm{M}, 2.9 \mathrm{mkat} / \mathrm{kg}, 0.6 / \mathrm{min}, 300 \mu \mathrm{M}, 22). When inactivated, the dioxygenase was found to contain per mole of active enzyme, (0.78 \mathrm{~mol}) of label from 3-thienyl(3\left[^{3} \mathrm{H}\right]) pyruvate and (0.85 \mathrm{~mol}) of label from 4-hydroxytetrafluorophenyl- (3\left[{ }^{3} \mathrm{H}\right]) pyruvate. The product formed from the enzyme-catalyzed oxidation of 3-thienylpyruvate was determined to be 3-carboxymethyl-3-thiolene-2-one. The implication of these results to the mechanism of the dioxygenase is considered. 1994 Academic Press, Inc.