Kinetic and mechanistic studies on the oxidation of the melilotate hydroxylase . 2-OH-cinnamate complex by molecular oxygen.

Abstract

The reaction mechanism of melilotate hydroxylase in the presence of 2-OH-cinnamate has been investigated by equilibrium titration, steady state kinetic and rapid reaction kinetic techniques. Analysis of the rapid reaction results for the reoxidation of reduced enzyme . 2-OH-cinnamate complex by molecular oxygen revealed the presence of an intermediate with visible absorption spectrum very similar to that of a C4a-flavin adduct. Repetition of this reoxidation experiment in the presence of 32 mM potassium iodide indicated the sequential appearance of two C4a-flavin adducts. Further analysis of these spectra indicated that hydroxylation of the substrate occurred during the conversion of the first intermediate into the second. Reaction of the oxidized enzyme . 2-OH-cinnamate complex with NADH was saturable in NADH. Oxidized enzyme was converted, in an apparent first order fashion, to a reduced enzyme . NAD charge-transfer-type species which subsequently decayed to reduced enzyme. The turnover number calculated from rapid reaction data was in good agreement with that determined from steady state analyses. Titration and rapid reaction results indicated that at no point in the reaction was there an observable intermediate which contained 2-OH-cinnamate in its phenolate form.