Transient-state intermediates involved in the hydride transfer step of the glutamate dehydrogenase reaction

Abstract

Stopped-flow spectrophotometric studies of the oxidative deamination of L-glutamate by L-glutamate dehydrogenase and TPN show that the rapid first phase or “burst” in absorbance is due to the formation of a previously unreported complex with a maximum absorbance at 332 mμ. The second slower phase consists of the maintenance of a constant level of this blue-shifted complex accompanied by a slow production of complex with a peak at about 348 mμ. Free TPNH release occurs only later as a third stage. Substitution of deuterium for the α-H atom of L-glutamate produces an isotope effect on the first slope of the reaction, but no effect on the burst height or later phases of the reaction. It is concluded that the burst phase represents the hydride transfer step of the reaction and that the 332 mμ peak is probably the resulting enzyme-TPNH-α-ketoglutarate-NH 4 + complex.