Mechanistic studies of the flavoprotein tryptophan 2-monooxygenase. 1. Kinetic mechanism.

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indole-3-acetamide, carbon dioxide, and water. The kinetic mechanism of the enzyme has been determined with tryptophan 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 rapid phase varies with the tryptophan concentration, with a limiting rate of 139 s-1 and an apparent Kd value of 0.11 mM. There is a primary deuterium kinetic isotope effect on the limiting rate of reduction of 2.4. The rapid phase is followed by a slow, concentration and isotope-independent phase that is much slower than turnover; this is ascribed to dissociation of a reduced enzyme-imino acid complex. In the absence of oxygen, tryptophan is converted to indolepyruvate imine. The rate of this reaction is the same as that of the rapid phase in the reduction. Reaction of the reduced enzyme-imino acid complex with oxygen to form oxidized flavin is monophasic, with a rate constant of 196 mM-1 s-1; no intermediates are detectable. The rate of formation of indole-3-acetamide agrees with the rate of reaction with oxygen. This is followed by slow product dissociation.