Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans.
The enzyme 4-hydroxyphenylacetate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating) (EC 1.14.13 ...; 4-hydroxyphenylacetate 1-monooxygenase; referred to here as 4-HPA 1-hydroxylase) was induced in Pseudomonas acidovorans when 4-hydroxyphenylacetate (4-PHA) was utilized as carbon source for growth; homogentisate and maleylacetoacetate were intermediates in the degradation of 4-HPA. A preparation of the hydroxylase that was free from homogentisate dioxygenase and could be stored at 4 C in the presence of dithioerythritol with little loss of activity was obtained by ultracentrifuging cell extracts; but when purified 18-fold by affinity chromatography the enzyme became unstable. Flavin adenine dinucleotide and Mg2+ ions were required for full activity. 4-HPA 1-hydrocylase was inhibited by KCl, which was uncompetitive with 4-HPA. Values of Ki determined for inhibitors competitive with 4-HPA were 17 muM dl-4-hydroxymandelic acid, 43 muM 3,4-dihydroxyphenylacetic acid, 87 muM 4-hydroxy-3-methylphenylacetic acid, and 440 muM 4-hydroxyphenylpropionic acid. Apparent Km values for substrates of 4-HPA 1-hydroxylase were 31 muM 4-HPA, 67 muM oxygen, 95 muM reduced nicotinamide adenine dinucleotide (NADH); AND 250 muM reduced nicotinamide adenine dinucleotide phosphate (NADPH). The same maximum velocity was given by NADH and NADPH. A chemical synthesis is described for 2-deutero-4-hydroxyphenylacetic acid. This compound was enzymatically hydroxylated with retention of half the deuterium in the homogentisic acid formed. Activity as substrate or inhibitor of 4-HPA 1-hydroxylase was shown only by those analogues of 4-HPA that possessed a hydroxyl group substituent at C-4 of the benze nucleus. A mechanism is suggested that accounts for this structural requirement and also for the observation that when 4-hydroxyphenoxyacetic acid was attacked by the enzyme, hydroquinone was formed by release of the side chain, probably as glycolic acid. Only one enantiometer of racemic 4-hydroxyhydratropic acid was attacked by 4-HPA 1-hydroxylase; the product, alpha-methylhomogentisic acid (2-(2,5-dihydroxyphenyl)-propionic acid), exhibited optical activity. This observation suggests that, during its shift from C-1 to C-2 of the nucleus, the side chain of the substrate remains bound to a site on the enzyme while a conformational change of the protein permits the necessary movement of the benzene ring.
D002474:Cell-Free System; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004226:Dithioerythritol; D004790:Enzyme Induction; D005978:Glutathione / Q000378:metabolism; D006713:Homogentisic Acid / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010618:Phenanthrolines / Q000378:metabolism; D010648:Phenylacetates / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology* / Q000378:metabolism; D014461:Ultracentrifugation
D010618:Phenanthrolines; D010648:Phenylacetates; D009243:NAD; D009249:NADP; D011189:Potassium Chloride; D004226:Dithioerythritol; D006899:Mixed Function Oxygenases; D005978:Glutathione; D008274:Magnesium; D006713:Homogentisic Acid