Interaction of bacterial luciferase with aldehyde substrates and inhibitors

Bacterial luciferase catalyzes the reaction of FMNH₂, O₂, and an aliphatic aldehyde to yield the carboxylic acid, FMN, water and blue-green light. The kinetics of the bacterial luciferase reaction were measured by stopped-flow spectrophotometry at pH 7 and 25 °C for the series of aldehydes from n-heptanal to n-undecanal. The rate of formation of the 4a- hydroperoxyflavin intermediate was dependent on the aldehyde concentration when mixtures of enzyme, FMNH₂, and aldehyde were rapidly mixed with O₂. At saturating aldehyde, the rate of formation of this intermediate was 100-fold slower than in the absence of aldehyde, demonstrating that an enzyme·FMNH₂·aldehyde complex can be formed. Numerical simulation of the time courses for these experiments supported the formation of this intermediate and its direct reaction with O₂. The kinetics of the light emitting reaction were dependent upon the chain length of the aldehyde substrate. Although the initial light intensity and the light emission decay rate were different for each aldehyde, the quantum yield for the reaction was independent of the aldehyde used. Luciferase was inhibited by high levels of the aldehyde substrate when the enzyme was assayed by mixing FMNH₂ with an aerobic mixture of enzyme and aldehyde. The extent of inhibition was dependent on the particular aldehyde used, and the binding affinity of the aldehyde for the free enzyme increased in parallel with the aldehyde chain length. The kinetics of the formation and decay of the various intermediates were also studied in the presence of n-alkylaldehyde analogs. These compounds decreased the rate of formation of the 4a-hydroperoxyflavin intermediate in much the same way as the aldehyde substrate, presumably by the formation of the enzyme·FMNH₂·analog ternary complex.