The reaction of the C30A mutant of trimethylamine dehydrogenase with diethylmethylamine

The role played by the 6-S-cysteinyl FMN bond of trimethylamine dehydrogenase in the reductive half-reaction of the enzyme has been studied by following the reaction of the slow substrate di ethyl methyl ami ne with a C30A mutant of the enzyme lacking the covalent flavin attachment to the polypeptide. Removal of the 6-S-cysteinyl FMN bond diminishes the limiting rate for the first of the three observed kinetic phases of the reaction by a factor of six. but has no effect on~the rate constants for the two subsequent kinetic phases. The flavin in the C30A enzyme recovered from the reaction of C30A enzyme with excess substrate is found to have been converted to the 6-hydroxy derivative, rendering the enzyme inactive. The noncovalently bound FMN of the C30A mutant enzyme is also converted to 6-hydroxyFMN and rendered inactive upon reduction with excess trimethylamine, but not by reduction with dithionite, even at high pH or in the presence of the effector tetramethylam monium chloride. These resuls demonstrate that one significant role of the 6-S-cysteinyl FMN bond is to prevent the inactivation of the enzyme during catalysis. A reaction mechanism is proposed whereby OH" attacks C(6) of a flavin-s übst r ate covalent adduct in the course of steady-state turnover to form 6-hydroxyFMN.