The nitrogen in amino acids, purines, pyrimidines, and other biomolecules ultimately comes from atmospheric nitrogen, N_2. The biosynthetic process starts with the reduction of N₂ to NH₃ (ammonia), a process called nitrogen fixation. Although higher organisms are unable to fix nitrogen, this conversion is carried out by some bacteria and archaea. Symbiotic Rhizobium bacteria invade the roots of leguminous plants and form root nodules in which they fix nitrogen, supplying both the bacteria and the plants. The amount of N₂ fixed by diazotrophic (nitrogen-fixing) microorganisms has been estimated to be 10¹¹ kilograms per year, about 60% of Earth's newly fixed nitrogen. Lightning and ultraviolet radiation fix another 15%; the other 25% is fixed by industrial processes. The industrial process for nitrogen fixation devised by Fritz Haber in 1910 is still being used in fertilizer factories.

The fixation of N₂ is typically carried out by mixing with H₂ gas over an iron catalyst at about 500°C and a pressure of 300 atmospheres. The extremely strong N≡N bond, which has a bond energy of 225 kcal mol^-1, is highly resistant to chemical attack. Indeed, Lavoisier named nitrogen gas “azote,” meaning “without life” because it is so unreactive. Nevertheless, the conversion of nitrogen and hydrogen to form ammonia is thermodynamically favorable; the reaction is difficult kinetically because intermediates along the reaction pathway are unstable.

To meet the kinetic challenge, the biological process of nitrogen fixation requires a complex enzyme with multiple redox centers. The nitrogenase complex, which carries out this fundamental transformation, consists of two proteins: a reductase, which provides electrons with high reducing power, and nitrogenase, which uses these electrons to reduce N₂ to NH_3. The transfer of electrons from the reductase to the nitrogenase component is coupled to the hydrolysis of ATP by the reductase. The nitrogenase complex is exquisitely sensitive to inactivation by O₂. Leguminous plants maintain a very low concentration of free O₂ in their root nodules by binding O₂ to leghemoglobin, a homolog of hemoglobin.

In principle, the reduction of N₂ to NH₃ is a six-electron process.

However, the biological reaction always generates at least 1 mol of H₂ in addition to 2 mol of NH₃ for each mole of N≡N. Hence, an input of two additional electrons is required.

In most nitrogen-fixing microorganisms, the eight high-potential electrons come from reduced ferredoxin, generated by photosynthesis or oxidative processes. Two molecules of ATP are hydrolyzed for each electron transferred. Thus, at least 16 molecules of ATP are hydrolyzed for each molecule of N₂ reduced.

Again, ATP hydrolysis is not required to make nitrogen reduction favorable thermodynamically. Rather, it is essential to reduce the heights of activation barriers along the reaction pathway, thus making the reaction kinetically feasible.

The next step in the assimilation of nitrogen into biomolecules is the entry of NH₄⁺ into amino acids. Glutamate and glutamine play pivotal roles in this regard. The α-amino group of most amino acids comes from the α-amino group of glutamate by transamination. Glutamine, the other major nitrogen donor, contributes its side-chain nitrogen atom in the biosynthesis of a wide range of important compounds, including the amino acids tryptophan and histidine.

Glutamate is synthesized from NH₄⁺ and α-ketoglutarate, a citric acid cycle intermediate, by the action of glutamate dehydrogenase. We have already encountered this enzyme in the degradation of amino acids. Recall that NAD⁺ is the oxidant in catabolism, whereas NADPH is the reductant in biosyntheses. Glutamate dehydrogenase is unusual in that it does not discriminate between NADH and NADPH, at least in some species.

A second ammonium ion is incorporated into glutamate to form glutamine by the action of glutamine synthetase. This amidation is driven by the hydrolysis of ATP. ATP participates directly in the reaction by phosphorylating the side chain of glutamate to form an acyl-phosphate intermediate, which then reacts with ammonia to form glutamine.

A high-affinity ammonia-binding site is formed only after the formation of the acyl-phosphate intermediate. A specific site for ammonia binding is required to prevent attack by water from hydrolyzing the intermediate and wasting a molecule of ATP. The regulation of glutamine synthetase plays a critical role in controlling nitrogen metabolism.

Glutamate dehydrogenase and glutamine synthetase are present in all organisms. Most prokaryotes also contain an evolutionarily unrelated enzyme, glutamate synthase, which catalyzes the reductive amination of α-ketoglutarate with the use of glutamine as the nitrogen donor.

The side-chain amide of glutamine is hydrolyzed to generate ammonia within the enzyme, a recurring theme throughout nitrogen metabolism. When NH₄⁺is limiting, most of the glutamate is made by the sequential action of glutamine synthetase and glutamate synthase. The sum of these reactions is

Note that this stoichiometry differs from that of the glutamate dehydrogenase reaction in that ATP is hydrolyzed. Why do prokaryotes sometimes use this more expensive pathway? The answer is that the value of K_M of glutamate dehydrogenase for NH₄⁺ is high ( ~ 1 mM), and so this enzyme is not saturated when NH₄⁺ is limiting. In contrast, glutamine synthetase has very high affinity for NH₄⁺. Thus, ATP hydrolysis is required to capture ammonia when it is scarce.

TRANSAMINATION

Aminotransferases catalyze the transfer of an α-amino group from an α-amino acid to an α-ketoacid. These enzymes, also called transaminases, generally funnel α-amino groups from a variety of amino acids to α-keto-glutarate for conversion into NH₄⁺.

Aspartate aminotransferase, one of the most important of these enzymes, catalyzes the transfer of the amino group of aspartate to α-ketoglutarate.

Alanine aminotransferase catalyzes the transfer of the amino group of alanine to α-ketoglutarate.

These transamination reactions are reversible and can thus be used to synthesize amino acids from α-ketoacids.