The Mechanism of Action of the Flavoprotein Melilotate Hydroxylase

Abstract

Abstract The reaction mechanism of melilotate hydroxylase has been investigated by a variety of kinetic methods. A steady state analysis has indicated that the enzyme has a mechanism involving two ternary complexes but not a quaternary complex of the enzyme and the three substrates. The reduction of the enzyme-melilotate complex by NADH has been studied by measuring fluorescence changes in the stopped flow apparatus. This reaction is second order and very rapid. The product of this reaction, observed by stopped-flow spectrophotometry, is believed to be a charge-transfer complex between the reduced FAD of the enzyme and NAD+. It has a broad, long wave length band centered at 750 nm and little absorption at 450 nm. This long wave length band disappears with a rate independent of the NADH concentration; this is presumably due to the dissociation of NAD+ from the complex. A charge-transfer complex can also be formed by anaerobic titration of reduced enzyme with NAD+ or 3-acetylpyridine-NAD+. The charge-transfer band is sensitive to the nature of the pyridine nucleotide. The spectrum of the complex between reduced enzyme and NAD+ appears to be identical with the spectrum observed transiently during the reduction of the enzyme by NADH in the stopped flow apparatus. The reaction of reduced melilotate hydroxylase with molecular oxygen in the absence of melilotate is a second order reaction. The rate of this reaction is enhanced more than 10-fold by the presence of melilotate, and the reaction profile becomes more complex. This complexity is due to the formation of an intermediate, which is believed to be an adduct of molecular oxygen to the reduced FAD of the enzyme. The spectrum of this intermediate has been determined by analog computer simulation using experimentally determined rate constants for its formation and decay, as well as the known extinction coefficients for reduced and oxidized enzyme. A reaction mechanism is proposed based on the steady state analysis. This analysis enabled the kinetic constants for the reaction to be determined. These kinetic constants have also been predicted from individually determined rate constants assuming the proposed mechanism. The values obtained by these two methods are in excellent agreement. They also correlate with the Kdiss previously determined for the dissociation of melilotate from the enzyme-substrate complex.