Phenol hydroxylase from yeast: a lysyl residue essential for binding of reduced nicotinamide adenine dinucleotide phosphate.

The inducible enzyme phenol hydroxylase from Trichosporon cutaneum is a FAD-containing monooxygenase which catalyzes the NADPH-dependent hydroxylation of phenol to catechol. The enzyme contains 16 cysteinyl residues, 6--8 of which are essential for retention of FAD and for activity. The complete amino acid composition is now reported as well as the results of studies with amino group reagents. A number of amino group reagents inhibit the enzyme severely, most of them with a concomitant, more or less extensive release of FAD. P-pyridoxal inhibits the enzyme specifically, without affecting its FAD content. The P-pyridoxal modified enzyme has a characteristic absorption peak at 325 nm indicating the presence of a N epsilon-pyridoxyllysyl derivative. Such a derivative was identified in hydrolysates of the modified enzyme by means of column chromatography. The results obtained with P-pyridoxal-modified enzyme indicate that a lysyl residue is essential for activity by being involved in binding of the co-substrate NADPH. These results are corroborated by kinetic studies showing competition between P-pyridoxal and NADPH for the binding site. The reactivity of the essential lysyl residue toward P-pyridoxal is significantly increased in the presence of phenol. Inhibition by excess phenol shifts toward lower concentrations in the presence of P-pyridoxal. On the basis of the present results together with previous findings, we propose that phenol acts as a substrate effector by causing a conformation change which exposes a reactive lysyl residue with a concomitant burying of the essential SH groups and a tighter attachment of FAD.