Interactions of substrate and non-substrate effectors with p-hydroxybenzoate hydroxylase from pseudomonas fluorescens

3,4-Dihydroxybenzoate (3,4-DOHB), 2,4-dihydroxybenzoate (2,4-DOHB), and benzoate facilitate the interaction of p-hydroxybenzoate hydroxylase with TPNH. The two dihydroxybenzoate effectors form 1:1 complexes with the enzyme, inducing large spectral perturbations and fluorescence quenching. The dissociation constants for 2,4-DOHB and 3,4-DOHB are 0.15 and 0.50 mM respectively. During the reaction of enzyme with TPNH and oxygen, all the 2,4-DOHB, <5% of the benzoate, and none of the 3,4-DOHB is hydroxylated.