Inhibition of p-hydroxybenzoate hydroxylase by anions: possible existence of two anion-binding sites in the site for reduced nicotinamide adenine dinucleotide phosphate.
Certain anions were found to inhibit p-hydroxybenzoate hydroxylase from Pseudomonas desmolytica. The inhibition was of competitive or mixed type with respect to NADPH (apparent Ki = 4-30 mM). Among the anions, monovalent anions such as halogen ions and azide inhibited ionization of the phenolic hydroxyl group of the substrate (p-hydroxybenzoate) on binding with the enzyme . substrate complex of p-hydroxybenzoate hydroxylase, without dissociating the substrate from the enzyme. On the other hand, multivalent anions (anions of polybasic acids), such as inorganic phosphate, borate, and sulfate, did not inhibit the ionization. Halogen ions induced remarkable spectral changes in the FAD moiety of the enzyme on binding, while the change due to inorganic phosphate was only slight. Chloride inhibited the binding of NADH with the enzyme as well as that of NADPH, whereas borate inhibited the binding of only NADPH. These results indicate that the monovalent and multivalent anions probably bind to the sites in the enzyme which interact, respectively, with the pyrophosphate and 2'-phosphate moieties of NADPH. The results provide strong support for the catalytic mechanism in which the phenolate anion of p-hydroxybenzoate participates in the process of substrate hydroxylation by C (4a) peroxyflavin. The results also suggest that repeated ionization/neutralization of the phenolic hydroxyl group of the substrate may occur during one cycle of the catalytic turnover.
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000037:antagonists & inhibitors*; D000838:Anions / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D002712:Chlorides / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D009249:NADP / Q000378:metabolism*; D013379:Substrate Specificity
D000838:Anions; D002712:Chlorides; D009249:NADP; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase