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While the air we breathe is rich in nitrogen gas (N2) and is the major component in air at 78%, this nitrogen is unavailable to us since nitrogen gas is very inert chemically. We want to briefly discuss how nitrogen gets assimilated, which refers to the formation of organic nitrogen molecules such as amino acids from inorganic nitrogen compounds in the environment. There are three steps involved in nitrogen assimilation.

Step 1 involves the fixation of nitrogen by bacteria that reside in soil and legume root nodules into usable forms such as ammonium, nitrites, and nitrates through the Nitrogen Cycle (Fig. 12-1). One thing that isn’t shown clearly in this figure is that ammonium is the endpoint of nitrogen fixation; nitrates and nitrites are reduced to ammonium, and it is ammonium that is used in step two of nitrogen assimilation. This is reflected in the overall reaction of nitrogen fixation:

N2 + 10 H+ + 16 ATP + 8 e- → 2 NH4+ + H2 + 16 ADP + 16 Pi

Note that there is an energy requirement for the fixation of nitrogen; it does not come cheap but it is an absolute necessity so that plants and subsequently animals can obtain the nitrogen in usable form.

Figure 12-1: The nitrogen cycle.

Source: http://www.ducksters.com/science/ecosystems/nitrogen_cycle.php Permission: This material has been reproduced in

accordance with the University of Saskatchewan interpretation of Sec.30.04 of the Copyright Act.

Step 2 of nitrogen assimilation into biomolecules occurs in plants and microorganisms, and involves the incorporation of ammonium into glutamate and glutamine. There are three enzyme-catalyzed reactions involved. The first reaction is catalyzed by glutamate dehydrogenase, which we studied in the previous module and noted that this was a potentially reversible reaction and that either NADH or NADPH could be used (Fig. 12-2). The reaction catalyzed is:

Figure 12-2: Glutamate dehydrogenase reaction is reversible and can be used to assimilate nitrogen.

Source: http://javed-abbas.tripod.com/notesonline/biochemistry/nitrogen-metabolism.html Permission: This material has been

reproduced in accordance with the University of Saskatchewan interpretation of Sec.30.04 of the Copyright Act.

Note that ammonium is used as a source of nitrogen to incorporate into an organic molecule i.e. assimilation of nitrogen.

The glutamate can then be used in a subsequent reaction to assimilate even more nitrogen; this is catalyzed by glutamine synthetase:

Glutamate + NH4+ + ATP → glutamine + ADP + Pi + H+

We’ll have more to say about this very important enzyme a bit later.

There is a third enzyme that is involved in the assimilation of nitrogen, although indirectly. Since there is a large need for glutamate because of the above reaction, there is a separate reaction that plants and microorganisms have that ensures that there are high levels of glutamate present. It is catalyzed by glutamate synthase:

α-ketoglutarate + glutamine + NADPH + H+ → 2 glutamate + NADP+

Step 3 in the assimilation of nitrogen involves using glutamate to donate its amino group to other α-ketoacids to form most of the other amino acids that are needed in the cell. This is done by the aminotransferases that we studies in the previous module. Recall that the reactions catalyzed by these enzymes are reversible; thus if glutamate levels are high, the reaction can flow in the opposite direction to that shown below in Fig. 12-3, producing the various amino acids.

Figure 12-3: The reaction catalyzed by aminotransferases. Note that this reaction can go in the reverse

direction depending on the relative concentrations of products and substrates. Thus, when glutamate

levels are high, it can transfer its amino group to otherα-ketoacids to form other amino acids.

Source: Nelson and Cox, Fig.18-4 in Lehninger Principles of Biochemistry, 5th edition. W.H. Freeman

and Company, 2008. Permission: This material has been reproduced in accordance with the University

of Saskatchewan Fair Dealing Guidelines, an interpretation of Sec.29.4 of the Copyright Act.

It should be noted that glutamine, and to a lesser extent glutamate, serve as a source of amino groups and nitrogen atoms in many biosynthetic processes. We will see some of these later in this module and in module 13.

Glutamine Synthetase Plays a Central Role in Nitrogen Metabolism

Glutamine synthetase is one of the major enzymes involved in assimilating nitrogen and is the primary regulatory point in nitrogen metabolism in most species. Not surprisingly, it is a ubiquitous enzyme, present in all organisms. Thus it is worth spending some time looking at this enzyme and its properties in detail. The overall reaction it catalyzes is shown below:

Glutamate + NH4+ + ATP → glutamine + ADP + Pi + H+

Note that ammonium is the source of the amino group that is incorporated into glutamine by this enzyme. It requires the cleavage of ATP to make this reaction energetically favourable. The glutamine formed can be incorporated into proteins, but it also serves as an important donor of amino groups for the synthesis of other biomolecules, which includes but is not limited to nucleotides and other amino acids.

The regulation of glutamine synthetase is highly complex and involves both covalent modification and allosteric regulation. There are a total of 8 allosteric inhibitors; all of them are nitrogen-containing biomolecules, 6 are molecules that glutamine is a provider of the nitrogen for their biosynthesis, and the other two are amino acids (Fig. 12-4). The degree of inhibition provided by these 8 inhibitors is more than additive, so that the cumulative effect of two, three or more is greater than the additive effect of each one individually. Another name for this type of inhibition where there is a greater than additive effect is concerted inhibition.

Figure 12-4: Allosteric inhibition of glutamine synthetase by 8 different effectors. Note that all of the

8 inhibitors are nitrogen-containing molecules. Source: Nelson and Cox, Fig. 22-6 in Lehninger Principles

of Biochemistry, 5th edition. W.H. Freeman and Company, 2008. Permission: This material has been

reproduced in accordance with the University of Saskatchewan Fair Dealing Guidelines, an interpretation

of Sec.29.4 of the Copyright Act.

Glutamine synthetase is also regulated by covalent modification, specifically by adenylylation (Fig. 12-5). Adenylylation refers to the linking of an AMP molecule to a tyrosine reside through the –OH group of tyrosine, forming an ester bond, using ATP as the donor of the AMP group.

Figure 12-5: Adenylylation of a substrate. ATP is the donor of the AMP group which is joined to an hydroxyl group, forming an ester bond. PPi is a product, which is

hydrolyzed to 2 Pi, which releases energy and drives the reaction forward. In the figure, the Acceptor is a protein and the –R group is a hydroxyl group.

Source: http://what-when-how.com/molecular-biology/adenylylation-molecular-biology/ Permission: This material has been reproduced in accordance with the

University of Saskatchewan interpretation of Sec.30.04 of the Copyright Act.

The quaternary structure of glutamine synthetase varies from species to species, but always involves multiple identical subunits that interact to form a multi-subunit enzyme. In many species including humans, it consists of 12 identical subunit arranged as two six-member “donuts” that sit on top of one another (Fig. 12-6). Each subunit has an active site. It is interesting to note that the tyrosine residue that becomes adenylylated in each subunit is located close to the active site.

Figure 12-6: The crystal structure of glutamine synthetase. Left hand panel: A side view showing the

two layers of subunits. Right hand panel: A top view showing the arrangement of the six identical

subunits in the top layer. The green dots are the location of the active site in each subunit.

Source: Nelson and Cox, Fig. 22-5a and 22-5b, respectively, in Lehninger Principles of Biochemistry,

5th edition. W.H. Freeman and Company, 2008. Permission: This material has been reproduced in

accordance with the University of Saskatchewan Fair Dealing Guidelines, an interpretation of

Sec.29.4 of the Copyright Act.

What is the consequence of adenylylation? It acts to sensitize the enzyme to the 8 allosteric inhibitors. In fact, research has shown that a glutamine synthetase that is completely devoid of any adenylylation is essentially insensitive to any of the allosteric inhibitors. Thus, a combination of covalent modification and allosteric inhibition is required to control the activity of this enzyme.