Inhibition of 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. strain P.J. 874 the enol tautomer of the substrate.
Progressive inactivation of purified 4-hydroxyphenylpyruvate dioxygenase (4-hydroxyphenylpyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating), EC 1.13.11.27) from Pseudomonas sp. strain P.J. 874 by enol-4-hydroxyphenylpyruvate was initially pseudo-first-order with respect to the remaining enzymic activity, as measured with an enol-borat assay at pH 7.5 and 37 degrees C. No inhibitory product was detected. Saturation kinetics suggests formation of a reversible complex prior to an inactivation event at the active site of the enzyme. The initial concentration of enol-4-hydroxyphenylpyruvate, which gave half-maximum inactivation, varied linearly with the assay concentration of ascorbate from 30 microM at zero (extrapolated value) to 0.8 mM at 20 mM ascorbate. The limiting rate constant for the inactivation increased linearly from 0.01 to 0.02 s-1 in this interval. Inhibition by ascorbate present during preincubations was partially relieved by enol-4-hydroxyphenylpyruvate. Inhibition by 1,2-dihydroxybenzene-3,5-disulfonic acid present during preincubations was prevented by ascorbate but not reversed by enol-4-hydroxyphenylpyruvate. The reductively-activated enzyme used keto-4-hydroxyphenylpyruvate as substrate for formation of 14CO2 and homogentisate. enol-4-Hydroxyphenylpyruvate was a noncompetitive inhibitor vs. keto-4-hydroxyphenylpyruvate with an intercept inhibition constant of about 40 microM when a 14CO2 assay was used. It is suggested that interaction of enol-4-hydroxyphenylpyruvate with enzyme-bound Fe3+, formed by autooxidation, caused the substrate inhibition of 4-hydroxyphenylpyruvate dioxygenase, long known to be relieved by a variety of reductants. The possible role for the inhibition mechanism in the regulation of tyrosine catabolism in vivo is discussed.
D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000037:antagonists & inhibitors*; D007536:Isomerism; D007700:Kinetics; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000037:antagonists & inhibitors*; D010667:Phenylpyruvic Acids / Q000494:pharmacology*; D011549:Pseudomonas / Q000201:enzymology; D013379:Substrate Specificity
D010667:Phenylpyruvic Acids; D010105:Oxygenases; D015111:4-Hydroxyphenylpyruvate Dioxygenase