On the mechanism of glutamine-dependent reductive amination of alpha-ketoglutarate catalyzed by glutamate synthase.

Highly purified preparations of glutamate synthase catalyze the TPNH-dependent reductive amination of cu-ketoglutarate to form L-glutamate in the presence of either L-glutamine or NH:,. Preparations of the enzyme that lack flavins (FAD and FMN) or the flavins and iron sulfide, catalyzed NH,,-mediated glutamate synthesis, but not glutamine-mediated glutamate synthesis. Participation of enzyme-flavin in the reductive amination reaction with glutamine as the nitrogen donor, but not with NH,, as the nitrogen donor, was also indicated by studies in which the enzyme was reduced with dithionite. Thus, incubation of the enzyme with Na,S,O,, cu-keto]‘4Clglutarate, and glutamine led to substantial formation of [‘“Clglutamate, which was shown to be of the L configuration; however, only trace amounts of [‘4C]glutamate were formed when glutamine was replaced by NH,,. Studies with stereospecifically labeled [:‘H]TPNH showed that glutamate synthase, like glutamate dehydrogenase, uses the hydrogen atom at the B side of C4 of the nicotinamide ring of TPNH. In studies with [“HITPNH (B) it was found that the products formed in the reductive amination of cY-ketoglutarate with glutamine and NH:, are different. Thus, in the reaction with glutamine, :‘H is transferred from [“HITPNH (B) to water; “H,O and glutamate are formed stoichiometrically. On the other hand, in the reaction with NH,, [“HIglutamate is formed. The enzyme also catalyzes rapid exchange of “H from [“HITPNH (B) with H,O, and a much slower exchange of :jH from [(Y“HIglutamate with water. The findings show that reductive amination with glutamine is flavin-mediated, whereas reductive amination with NH, is not and therefore closely resembles the reaction catalyzed by glutamate dehydrogenase in which :‘H is transferred from [:‘H]TPNH (B) to [‘lH]glutamate. The findings are consistent with the presence in highly purified glutamate synthase preparations (from Ewcherichia coli and Aerohacter aerogenes) of two different catalytic entities. The pH dependence of the glutaminase activity of glutamate synthase was determined; this activity, like the glutaminase activity of carbamyl phosphate synthetase, is enhanced by storage of the enzyme at pH 9.