DEUTERIUM KINETIC ISOTOPE EFFECTS AND THE MECHANISM OF THE BACTERIAL LUCIFERASE REACTION

A combined experimental and theoretical investigation of the deuterium isotope effects on the bacterial luciferase reaction is described. Th e experimental studies focus on determining if the unusual aldehydic d euterium isotope effect of similar to 1.5 observed in these reactions is an intrinsic isotope effect resulting from a single rate-limiting s tep or is a composite of multiple rate-limiting steps, The isotope eff ect observed is not significantly affected by variation in the aldehyd e chain length, changes in the pH over a range of 6-9, use of alpha C1 0GA and alpha C106S site-directed mutants, or chloride substitution at the 8-position of the reduced flavin, though the isotope effect is de creased when the X-methoxy-substituted flavin is used as a substrate. From these observations it is concluded that the aldehydic isotope eff ect arises from the change in rate of a single kinetic step, A stopped -flow kinetic analysis of the microscopic rate constants for the react ions of 1-[H-1]decanal and 1-[H-2]decanal in the bacterial luciferase reaction was carried out, and aldehyde hydration isotope effects were determined, From the results it is estimated that the aldehydic deuter ium isotope effect is similar to 1.9 after formation of an intermediat e flavin C4a-hydroperoxy hemiacetal, Ab initio calculations were used to examine The transformation of the aldehyde into a carboxylic acid a nd to predict isotope effects for possible mechanisms. These calculati ons indicate that the mechanism involving rate-limiting electron trans fer from the flavin C4-a-hydroxide to an intermediate dioxirane is con sistent with the enigmatic aldehydic Isotope effect and that the inter mediacy of a dioxirane is energetically plausible.