Alternate substrates and inhibitors of bacterial 4-hydroxyphenylpyruvate dioxygenase

A variety of analogues of (4-hydroxyphenyl)pyruvic acid were synthesized, and the reactions of these compounds with the 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874 were examined. Several of the ring-substituted substrate analogues are reversible inhibitors of the enzyme, the most potent being the competitive inhibitor (2,6-difluoro-4-hydroxyphenyl) pyruvate (Ki = 1.3 microM). Two substrate analogues (2-fluoro-4-hydroxyphenyl)pyruvate and [(4-hydroxyphenyl)thio]pyruvate proved to be alternate substrates for the enzyme. The former compound is converted to (3-fluoro-2,5-dihydroxyphenyl)acetate in an essentially normal catalytic sequence including oxidative decarboxylation, ring hydroxylation, and side-chain migration. The latter compound, however, undergoes oxidative decarboxylation and sulfoxidation to give [(4-hydroxyphenyl)sulfinyl]acetate; ring oxidation is not observed. The implications of these results with regard to the catalytic mechanism of 4-hydroxyphenylpyruvate dioxygenase are discussed.