Biochemistry - The Chemical Reactions of Living Cells, Volume 3 - D. Metzler 1980

Metabolism of Nitrogen-Containing Compounds
Incorporation of NH3 into Amino Acids and Proteins
Glutamate Dehydrogenase and Glutamate Synthase

According to current models, the reaction catalyzed by Glutamate dehydrogenase (Fig. 14-2, stage a; see also Chapter 8, Sections 3, 4) represents the primary pathway for the reversible incorporation of ammonia into glutamic acid. Either NADH or NADPH can serve as the reducing agent in this reaction. In Eukaryotic Cells, glutamate dehydrogenase is located predominantly in the Cell/35.html">Mitochondria. Subsequently, through the action of transaminases both inside and outside the mitochondria, the nitrogen of glutamic acid is redistributed and incorporated into Other Amino Acids. Aspartate aminotransferase (Chapter 8, Sections D, 3 and D, 7) is particularly active, maintaining equilibrium between aspartate and oxaloacetate on the one hand, and the $\alpha$-ketoglutarate/glutamate pair on the other.

In E. coli and many other Bacteria, the reductive amination of $\alpha$-ketoglutarate is carried out by glutamate synthase (Fig. 14-2, stage b). In this reaction, nitrogen is supplied by the amide group of glutamine. There is every reason to believe that ammonia is released from this group within the Active Site of the enzyme. Consequently, The formation of a Schiff base and reduction by NADPH can proceed in exactly the same manner as in reaction a (Chapter 8, Sections 3, 4). The difference is that one of the two glutamate molecules formed in reaction b must be converted back into glutamine by the action of glutamine synthase (Section B, 2) utilizing one molecule of ATP.

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FIG. 14-2. Biosynthesis of glutamic acid, glutamine, Proline, and Lysine from $\alpha$-ketoglutarate.

As a result of this coupling of the reaction with ATP Hydrolysis, the equilibrium in reaction b is strongly shifted toward glutamate synthesis. Glutamate synthase is characterized by a very low $K_m$ value for glutamine (and, consequently, for its amide nitrogen).

E. coli glutamate synthase is a large protein with a Molecular Weight of 800,000, containing flavin, iron, and $S^{2-}$ in a ratio of 1:4:4 [18]. The enzyme uses NADH as a reducing agent; however, some experiments indicate that the tightly bound flavin, once reduced, serves as the direct donor of electrons required for the reduction of the Schiff base formed by $\alpha$-ketoglutarate with ammonia [18]. It remains unclear precisely what Functions the flavin and iron-sulfur prosthetic groups serve. The iron-sulfur group may provide coupling not only with NADPH, but also with reduced ferredoxin.

It has recently been discovered that glutamate synthase appears to provide the main pathway for nitrogen incorporation into amino acids in Yeast [19] and higher green plants. In the latter case, reduced ferredoxin can serve as the reducing agent [20].