Mechanism of Action of p-Hydroxybenzoate Hydroxylase from Pseudomonas putida

The mechanism of action of p-hydroxybenzoate hydroxylase from Pseudomonas putida, strain M-6, has been investigated. The aromatic substrate analogues, benzoate, p-fluorobenzoate, p-chlorobenzoate, p-nitrobenzoate, p-aminobenzoate, and 6-hydroxynicotinate, are found to be competitive inhibitors. This finding differs from the previously reported noncompetitive behavior in a different buffer system. The optical activity of the enzyme-inhibitor complex has been studied. From the kinetic and circular dichroism (CD) measurements, we have found that the carboxyl moiety is necessary and sufficient for the enzyme-substrate binding, whereas the hydroxyl group alone will not lead to binding. There are two classes of inhibitory analogues: one causes changes in CD spectra of the enzyme similar to those evoked by the substrate, and the other does not cause significant changes. The results indicate that more than one mode of enzyme-inhibitor interaction is involved. The CD of the enzyme-NADPH complex under anaerobic conditions suggests that the oxidized enzyme and reduced pyridine nucleotide form a complex, both in the absence and presence of the substrate, p-hydroxybenzoate. Furthermore, evidence for a ternary complex is given.