Allosteric Regulation

Chapter 22 Biosynthesis of Amino Acids, Nucleotides, and Related Molecules assimilation of NH4 into glutamate requires two reactions. First, glutamine synthetase catalyzes the reaction of glutamate and NH4 to yield glutamine. This reaction takes place in two steps, with enzyme-bound y-glutamyl phosphate as an intermediate (see Fig. 18-8):

(1) Glutamate + ATP-> 7-glutamyl phosphate + ADP

(2) y-Glutamyl phosphate + NH4 -> glutamine + Pi + H+

Glutamine synthetase is found in all organisms. In addition to its importance for NH4 assimilation in bacteria, it has a central role in amino acid metabolism in mammals, converting toxic free NH4 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. a-Ketoglutarate, an intermediate of the citric acid cycle, undergoes reductive amination with glutamine as nitrogen donor:

a-Ketoglutarate 4 glutamine 4 NADPH 4 H4-

2 glutamate 4 NADP4

The net reaction of glutamine synthetase and glutamate synthase (Eqns 22-1 and 22-2) is a-Ketoglutarate 4 NH4 4 NADPH 4 ATP->

L-glutamate 4 NADP+ 4 ADP 4 P;

Glutamate synthase is not present in animals, which, instead, maintain high levels of glutamate by processes such as the transamination of a-ketoglutarate during amino acid catabolism.

Glutamate can also be formed in yet another, albeit minor, pathway: the reaction of a-ketoglutarate and NH4 to form glutamate in one step. This is catalyzed by l-glutamate dehydrogenase, an enzyme present in all organisms. Reducing power is furnished by NADPH:

a-Ketoglutarate 4 NH4 4 NADPH->

L-glutamate 4 NADP4 4 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 NH4 (~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 NH44 destined for the urea cycle.) Concentrations of NH44 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).

Glutamine Synthetase Is a Primary Regulatory Point in Nitrogen Metabolism

The activity of glutamine synthetase is regulated in virtually all organisms—not surprising, given its central metabolic role as an entry point for reduced nitrogen. In enteric bacteria such as E. coli, the regulation is unusually complex. The enzyme has 12 identical subunits of Mr 50,000 (Fig. 22-5) and is regulated both alloster-ically and by covalent modification. Alanine, glycine, and at least six end products of glutamine metabolism are allosteric inhibitors of the enzyme (Fig. 22-6). Each inhibitor alone produces only partial inhibition, but the effects of multiple inhibitors are more than additive, and all eight together virtually shut down the enzyme. This control mechanism provides a constant adjustment of glutamine levels to match immediate metabolic requirements.

Allosteric Regulation

FIGURE 22-5 Subunit structure of glutamine synthetase as determined by x-ray diffraction. (PDB ID 2GLS) (a) Side view. The 12 sub-units are identical; they are differently colored to illustrate packing and placement. (b) Top view, showing active sites (green).

FIGURE 22-5 Subunit structure of glutamine synthetase as determined by x-ray diffraction. (PDB ID 2GLS) (a) Side view. The 12 sub-units are identical; they are differently colored to illustrate packing and placement. (b) Top view, showing active sites (green).

Superimposed on the allosteric regulation is inhibition by adenylylation of (addition of AMP to) Tyr397, located near the enzyme's active site (Fig. 22-7). This co-valent modification increases sensitivity to the allosteric inhibitors, and activity decreases as more subunits are adenylylated. Both adenylylation and deadenylylation are promoted by adenylyltransferase (AT in Fig. 22-7), part of a complex enzymatic cascade that responds to levels of glutamine, a-ketoglutarate, ATP, and Pi. The activity of adenylyltransferase is modulated by binding to a regulatory protein called Pn, and the activity of Pjj, in turn, is regulated by covalent modification (uridylylation), again at a Tyr residue. The adenylyltransferase complex with uridylylated Pn (Pn-UMP) stimulates deadenylylation, whereas the same complex

FIGURE 22-6 Allosteric regulation of glutamine synthetase. The enzyme undergoes cumulative regulation by six end products of glutamine metabolism. Alanine and glycine probably serve as indicators of the general status of amino acid metabolism in the cell.

Glutamate

Glutamate

Adenylyltransferase Glutamine

M FIGURE 22-7 Second level of regulation of glutamine synthetase: covalent modifications. (a) An adenylylated Tyr residue. (b) Cascade leading to adenylylation (inactivation) of glutamine synthetase. AT represents adenylyltransferase; UT, uridylyltransferase. Details of this cascade are discussed in the text.

M FIGURE 22-7 Second level of regulation of glutamine synthetase: covalent modifications. (a) An adenylylated Tyr residue. (b) Cascade leading to adenylylation (inactivation) of glutamine synthetase. AT represents adenylyltransferase; UT, uridylyltransferase. Details of this cascade are discussed in the text.

Adenylyltransferase Glutamine

FIGURE 22-6 Allosteric regulation of glutamine synthetase. The enzyme undergoes cumulative regulation by six end products of glutamine metabolism. Alanine and glycine probably serve as indicators of the general status of amino acid metabolism in the cell.

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