On the stable enzyme-substrate complex of p-hydroxybenzoate hydroxylase. Evidences for the proton uptake from the substrate.

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas desmolytica is known to form a stable complex with its substrate. Investigations were made on properties of the complex by spectrophotometric measurements and photooxidation. The addition ofp-hydroxybenzoate or benzoate to the oxidized (with respect to bound FAD) enzyme solution caused changes in absorption, fluorescence, and CD spectra, most of which could be ascribed to spectral changes of the enzyme by the complex formation. Among them, the change in the absorbance around 285 nm caused by p-hydroxybenzoate was pH-dependent with an enormous increase in the alkaline side, indicating the formation of the phenolate anion of the substrate above pH 7 (pK = 7.13,20%). On the other hand, the change caused by benzoate was smaller and pHindependent. p-Hydroxybenzoate hydroxylase was inactivated by the photooxidation mediated by methylene blue, following pseudo-first order kinetics. The pH dependency of the inactivation rate showed the involvement of an ionizing group with a pK of 7.02 (25°C) in the inactivation. The addition of p-hydroxybenzoate shifted the pK toward the alkaline side by more than 1 pH unit (to 8.20), while benzoate exerted little effect. The pH dependency of the apparent dissociation constant of the enzyme l p-hydroxybenzoate complex also revealed a minor change around pH 7. These results strongly suggest that an ionizing group with a pK of 7.0 (25”C, in free enzyme) participates in the enzyme l substrate complex formation of p-hydroxybenzoate hydroxylase, having an interaction with the hydroxyl group of the substrate. The ionizing group is judged to be most likely an imidazole of histidine, from the pK value and other evidences. The results also suggest that the proton uptake from the substrate occurs by the complex formation in the alkaline side resulting in the formation of a phenolate anion of the substrate and the protonation of the ionizing group. Discussion was made on the phenomenon together with its effects on the following steps of the enzymatic reaction, i.e. reactions with NADPH and molecular oxygen.