MECHANISTIC STUDIES OF THE FLAVOPROTEIN TRYPTOPHAN 2-MONOOXYGENASE .1. KINETIC MECHANISM

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative de carboxylation of tryptophan to indole-3-acetamide, carbon dioxide, and water. The kinetic mechanism of the enzyme has been determined with t ryptophan as substrate at pH 8.3. Initial velocity patterns, when both amino acid and oxygen concentrations are varied, are sequential with tryptophan and ping-pong with phenylalanine and methionine. Reduction by tryptophan in the absence of oxygen is biphasic. The rate of the ra pid phase varies with the tryptophan concentration, with a limiting ra te of 139 s(-1) and an apparent K-d value of 0.11 mM. There is a prima ry deuterium kinetic isotope effect on the limiting rate of reduction of 2.4. The rapid phase is followed by a slow, concentration and isoto pe-independent phase that is much slower than turnover; this is ascrib ed to dissociation of a reduced enzyme-imino acid complex. In the abse nce of oxygen, tryptophan is converted to indolepyruvate imine. The ra te of this reaction is the same as that of the rapid phase in the redu ction. Reaction of the reduced enzyme-imino acid complex with oxygen t o form oxidized flavin is monophasic, with a rate constant of 196 mM(- 1) s(-1); no intermediates are detectable. The rate of formation of in dole-3-acetamide agrees with the rate of reaction with oxygen. This is followed by slow product dissociation.