A non-substrate effector of p-hydroxybenzoate hydroxylase

Abstract

A substrate analogue, 6-hydroxynicotinate (6-OHNA), facilitates reduction of the enzyme bound flavin by TPNH but is not hydroxylated in the reoxidation of the enzyme by oxygen. Binding of 6-OHNA to oxidized p-hydroxybenzoate hydroxylase is accompanied by a perturbation of the visible spectrum and enhancement of the flavin fluorescence. Titrations using these properties indicate the formation of a 1:1 complex with a dissociation constant of 2.5 x 10-4 M. At high concentrations of 6-OHNA, binding of 6-OHNA to additional site(s) has been observed. The 6-OHNA binding is competitive with p-hydroxybenzoate binding as indicated by the reversal of p-hydroxybenzoate quenching of flavin fluorescence on addition of 6-OHNA. The presence of the effector, 6-OHNA, is necessary for the catalytic oxidation of TPNH.