Phenol hydroxylase from yeast. A model for phenol binding and an improved purification procedure.

The binding of phenol to phenol hydroxylase was studied by equilibrium dialysis, spectrophotometric titration and by steady-state kinetics. A binding model with two identical, negatively cooperative, effector/substrate-binding sites per enzyme dimer is proposed. The spectral perturbation caused by phenol and the kinetics of the overall reaction were analysed with relation to the enzyme-phenol complexes of the binding model. The main part of the spectral perturbation as well as of the increase in NADPH oxidation rate was achieved by one molecule of phenol bound per enzyme dimer. The properties of different enzyme-phenol complexes, in terms of spectral changes, hydroxylase activity, oxidase activity and substrate inhibition are discussed. A new purification procedure is described.