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

Shoun, H.; Beppu, T.; Arima, K.

Journal of Biological Chemistry 254(3): 899-904


ISSN/ISBN: 0021-9258
PMID: 33179
Accession: 068518558

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p-Hydroxybenzoate hydroxylase (EC from P. desmolytica 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.degree. C). The change caused by benzoate was smaller and pH-independent. p-Hydroxybenzoate hydroxylase was inactivated by the photooxidation mediated by methylene blue, following pseudo-1st order kinetics. The pH dependency of the inactivation rate showed the involvement of an ionizing group with a pK of 7.02 (25.degree. 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 Kd constant of the enzyme.cntdot.p-hydroxybenzoate complex also revealed a minor change around pH 7. An ionizing group with a pK of 7.0 (25.degree. C, in free enzyme) apparently participates in the enzyme.cntdot.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 proton uptake from the substrate apparently 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 O2.