Suicide inactivation of the flavoenzyme D-lactate dehydrogenase by alpha-hydroxybutynoate

Abstract

The acetylenic α-hydroxy acid 2-hydroxy-3- butynoate (αHB) is a substrate and an irreversible inactivator of the FAD-containing flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii. On the average, the enzyme undergoes five catalytic turnovers with αHB in air at pH 7.0 before being inactivated. Irreversible inactivation is due to the conversion of the flavin to a pink adduct with visible absorption peaks at 522, 382, and 330 nm and weak fluorescence with an emission maximum at 635 nm. The adduct is stable and can be released from the enzyme and purified. It retains a structure analogous to FAD since it binds to the FAD-specific apo-D-amino acid oxidase. It can be further converted to an FMN analogue with phosphodiesterase which binds to the FMN-specific apoflavodoxin. Experiments were conducted to test whether inactivation was initiated by an αHB allene carbanion or the dehydrogenation product of αHB. Kinetic studies proved inconclusive in that a rapid equilibrium between an oxidized enzyme-allene carbanion pair and reduced enzyme-keto acid pair would make these two species kinetically equivalent. The olefinic substrate 2- hydroxy-3-butenoate, however, produced no flavin adduct. Since the keto acid derived from the oxidation of this αhydroxy acid is expected to be as reactive as 2-keto-3-butynoate, it is concluded that an allene carbanion produced by abstraction of the α-hydrogen of αHB is the reactive species which covalently adds to the flavin.