PH-dependent studies reveal an efficient hydroxylation mechanism of the oxygenase component of p-hydroxyphenylacetate 3-hydroxylase.

ABSTRACT: p-Hydroxyphenylacetate (HPA) 3-hydroxylase (HPAH) catalyzes the hydroxylation of HPA at the ortho-position to yield 3,4-dihydroxyphenylacetate. The enzyme is a flavin-dependent two-component monooxygenase that consists of a reductase component and an oxygenase component (C(2)). C(2) catalyzes the hydroxylation of HPA using oxygen and reduced FMN as co-substrates. To date, the effects of pH on the oxygenation of the two-component monooxygenases have never been reported. Here, we report the reaction kinetics of C(2)·FMNH(-) with oxygen at various pH values investigated by stopped-flow and rapid quenched-flow techniques. In the absence of HPA, the rate constant for the formation of C4a-hydroperoxy-FMN (∼1.1 × 10(6) m(-1)s(-1)) was unaffected at pH 6.2-9.9, which indicated that the pK(a) of th

SUBMITTER: Ruangchan N 

PROVIDER: S-EPMC3012978 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature