Purification and some properties of human 4-hydroxyphenylpyruvate dioxygenase (I).

4-Hydroxyphenylpyruvate dioxygenase (I-hydroxyphenylpyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating), EC 1.13.11.27) has been purified 800-fold in 25% yield from human liver by a procedure involving ammonium sulfate fractionation of an acetone powder extract and chromatography on hydroxylapatite, sulfopropyl-Sephadex C-50, and triethylaminoethyl cellulose. The preparation obtained was homogenous as determined by electrophoresis, gel filtration, sedimentation equilibrium, and immunodiffusion. A molecular mass of 87 kilodaltons was obtained by sedimentation equilibrium. A subunit mass of 43 kilodaltons was obtained by electrophoresis in polyacrylamide gel containing sodium dodecyl sulfate. The enzymic activity had two pH optima around 4.5 and 7.8. The enzyme was equally activated by ascorbate or by a combination of 2,6-dichlorophenolindophenol and glutathione in the presence of catalase. It was very sensitive to H,O, inhibition, which was abolished by a reductant and catalase. Addition of Fez+ and of various metal salts had no stimulatory effect on enzyme activity. The activity with the keto form of 4-hydroxyphenylpyruvate was 40 times higher than with enol tautomer as substrate. During optimized assay conditions, the apparent Michaelis constants were about 50 FM for 02, 0.03 mM for 4-hydroxyphenylpyruvate, and 0.05 mM for phenylpyruvate. The corresponding apparent maximal velocities were 4 and 0.02 mmol mine1 (g of protein)-‘. The enzyme activity was strongly inhibited by iron and copper chelators. The activity of enzyme inhibited by diethyldithiocarbamate was restored by dialysis. Enzyme inhibited by bathophenanthroline was reactivated to -30% by dialysis and to -60% by addition of Fe’+. 4-Hydroxyphenylpyruvate dioxygenase activity was not inhibited by different thiol group reagents in 1 mM concentrations with the exception of mercurials, which inhibited in lower concentrations. The enzyme contained about five thiol groups and one disulfide bridge/87 kilodaltons as determined by titration with 4-ehloromercuribenzoate and 5,5’dithiobis(2-nitrobenzoate) and by alkylation with iodo[lWlacetate and iodo[l-14Clacetamide.