Overview of Nitrogen Metabolism:- Ammonia Is Incorporated into Biomolecules through Glutamate and Glutamine
Reduced nitrogen in the form of NH4 is assimilated into amino acids and then into other nitrogen-containing bio molecules. Two amino acids, glutamate and glutamine, provide the critical entry point. Recall that these same two amino acids play central roles in the catabolism of ammonia and amino groups in amino acid oxidation. Glutamate is the source of amino groups for most other amino acids, through transamination reactions (the reverse of the reaction shown in Fig. 18–4). The amide nitrogen of glutamine is a source of amino groups in a wide range of biosynthetic processes. In most types of cells, and in extracellular fluids in higher organisms, one or both of these amino acids are present at higher concentrations—sometimes an order of magnitude or more higher—than other amino acids. An Escherichia coli cell requires so much glutamate that this amino acid is one of the primary solutes in the cytosol. Its concentration is regulated not only in response to the cell’s nitrogen requirements but also to maintain an osmotic balance between the cytosol and the external medium. The biosynthetic pathways to glutamate and glutamine are simple, and all or some of the steps occur in most organisms. The most important pathway for the assimilation of NH+4 into glutamate requires two reactions. First, glutamine synthetase catalyzes the reaction of glutamate and NH+4 to yield glutamine. This reaction takes place in two steps, with enzyme-bound γ-glutamyl phosphate as an intermediate (see Fig. 18–8):
(1) Glutamate + ATP→ γ-glutamyl phosphate + ADP
(2) γ- Glutamyl phosphate+ NH+4→ glutamine +Pi+H+
Sum: Glutamate+NH+4+ATP→glutamine+ADP+Pi+H+(22–1)
Glutamine synthetase is found in all organisms. In addition to its importance for NH+4 assimilation in bacteria, it has a central role in amino acid metabolism in mammals, converting toxic free NH+4 to glutamine for transport in the blood (Chapter 18). In bacteria and plants, glutamate is produced from glutamine in a reaction catalyzed by glutamate synthase. α-Ketoglutarate, an intermediate of the citric acid cycle, undergoes reductive amination with glutamine as nitrogen donor:
α-Ketoglutarate+ α-Ketoglutarate + NADPH+H+→2 glutamate + NADP+ (22–2)
The net reaction of glutamine synthetase and glutamate synthase (Eqns 22–1 and 22–2) is
α-Ketoglutarate + NH4++NADPH+ATP→L-glutamate+ NADP++ADP+Pi
Glutamate synthase is not present in animals, which, instead, maintain high levels of glutamate by processes such as the transamination of -ketoglutarate during amino acid catabolism. Glutamate can also be formed in yet another, albeit minor, pathway: the reaction of α-ketoglutarate and NH4 to form glutamate in one step. This is catalyzed by L-glutamate dehydrogenase, an enzyme present in all or ganisms. Reducing power is furnished by NADPH:
α-Ketoglutarate + NH+4 + NADPH → L-glutamate + NADP+ + H2O
We encountered this reaction in the catabolism of amino acids (see Fig. 18–7). In eukaryotic cells, L-glutamate dehydrogenase is located in the mitochondrial matrix. The reaction equilibrium favors reactants, and the Km for NH+4 (~1 mM) is so high that the reaction probably makes only a modest contribution to NH4 assimilation into amino acids and other metabolites. (Recall that the glutamate dehydrogenase reaction, in reverse (see Fig. 18–10), is one source of NH+4 destined for the urea cycle.) Concentrations of NH+4 high enough for the glutamate dehydrogenase reaction to make a significant contribution to glutamate levels generally occur only when NH3 is added to the soil or when organisms are grown in a laboratory in the presence of high NH3 concentrations. In general, soil bacteria and plants rely on the two-enzyme pathway outlined above (Eqns 22–1, 22–2).
