Copyright © 2000-2003 Mark Brandt, Ph.D. 24Introduction to amino acid metabolismOverviewThe body has a small pool of free amino acids. The pool is dynamic, and isconstantly being used as a source of substrate for various reactions, and isconstantly being replenished. Free amino acids are not stored, except as part oflarger molecules (i.e. proteins).Amino acids can be used for a variety of functions. The primary function of aminoacids is to act as the monomer unit in protein synthesis. Amino acids can be alsoused as substrates for biosynthetic reactions; the nucleotide bases, heme, and anumber of hormones and neurotransmitters are derived from amino acids. Finally,the carbon skeleton of all of the amino acids broken down for energy.Nitrogen metabolismUnlike glucose or fatty acids, amino acids contain nitrogen. Biologically relevantinorganic nitrogen molecules include dinitrogen (N2), nitrogen oxides (includingNO2– and NO3–), and ammonium (NH4+)4.Ammonium is the most useful form of inorganic nitrogen in most organisms,especially in animals. Unfortunately ammonium is toxic to animals. The reasons forthis toxicity are incompletely understood, but most symptoms involve altered brainfunction, suggesting that the central nervous system is vulnerable to ammonium-induced damage. Ammonium therefore must be handled carefully, and physiologicalnitrogen metabolism must take this into account. 4 Ammonium is the protonated form of ammonia (NH3); ammonium is the major species in aqueoussolution.Copyright © 2000-2003 Mark Brandt, Ph.D. 25Ammonium can come from several sources depending on the organism: 1) organicnitrogen: nitrogen attached to organic molecules that can be metabolized; 2) freeammonium; 3) nitrogen oxides (especially nitrate); and 4) dinitrogen.Nitrogen fixationN2 is inaccessible to most organisms, because of the strong bond between thenitrogen atoms. Although the conversion of N2 to NH3 has a ∆G° of about –33kJ/mol, the activation energy barrier for the reduction of N2 is very large.A few bacteria are capable of reducing dinitrogen to ammonia. These are callednitrogen-fixing bacteria; some are free living, but many are symbiotes of plants,especially legumes such as soybeans, peas and alfalfa.The nitrogen fixation reaction requires specialized proteins, the products of the nifgenes, which code for nitrogenase and its accessory proteins. Nitrogenase requiresiron, sulfur, and molybdenum as cofactors. Nitrogenase is rapidly denatured byoxygen, and therefore requires an oxygen-free environment.Legumes have leghemoglobin, a monomeric globin with high oxygen affinity. Itfunctions to protect the bacteria from free oxygen, by transferring oxygen only tothe bacterial cytochrome c oxidase. Free-living bacteria either live in anaerobicenvironments, or use uncoupling agents to increase their rate of oxygen reduction toprotect their nitrogenase complex.The nitrogen fixation reaction is expensive: at least 16 ATP are required toovercome the energy barrier in dinitrogen. The actual energy requirements areusually higher than the minimum stoichiometry shown below due to wasteful sidereactions. This means that organisms capable of fixing nitrogen have considerableenergy requirements. Legumes use ~20% of their ATP production to supply energyfor their symbiotic bacteria.The nitrogen fixation process requires electrons. In free living cyanobacteria, theelectrons are derived from a photosynthetic electron transport chain. The symbioticnitrogen-fixing bacteria of legumes are present in root nodules; because they are notexposed to sunlight, these bacteria must use electrons from metabolic sources (suchas the pyruvate dehydrogenase reaction) to drive nitrogen reduction.Humans can only perform N2 reduction using technological assistance. The Haberprocess, invented shortly before World War I, uses high pressures of hydrogen gas(200 atmospheres) and temperatures (700 K) to achieve what the bacteria manageat ambient temperature and pressure.Copyright © 2000-2003 Mark Brandt, Ph.D. 26Nitrogen assimilationPlants can use either ammonium or nitrogen oxides (especially nitrate) as sources ofusable nitrogen. Nitrate is formed by microorganisms that can use ammonium as anenergy source, and is thus the lowest energy form of nitrogen. On the other hand,nitrate and other nitrogen oxides are major components in explosives, which is whyfertilizer can be dangerous. The explosive potential of the common fertilizerNH4NO3 when combined with readily available carbon compounds such as diesel oilhas led to some limits on the sale of this material.Nitrate reduction requires electrons, derived from photosynthesis, to produceammonium. The reduction of nitrate must be followed by ammonium fixation, theprocess of attaching ammonium ions to carbon compounds. The reactions used forthis purpose are discussed below.Unlike plants, animals use organic nitrogen derived from their diet for essentiallyall of their nitrogen requirements. Animals require nitrogen in reduced form andrelease most nitrogen in reduced form; in general, animals cannot reduce nitrogenoxides, and do not excrete these compounds.Most organisms have three major reactions that incorporate inorganic nitrogen intoorganic compounds. These reactions are catalyzed by glutamate dehydrogenase,glutamine synthetase, and one isozyme of carbamoyl phosphate synthase (this lastenzyme we will discuss later, during the discussion of the urea cycle).In addition, one pathway for glycine synthesis uses inorganic ammonium; undermost conditions, this reaction is a relatively minor ammonium fixation reaction.Finally, some microorganisms can fix ammonium using asparagine synthetase,although higher organisms use glutamine as the ammonium donor for this reaction.Glutamate dehydrogenase uses reducing equivalents from NADPH to bindammonium to a-ketoglutarate. It can also catalyze the reverse reaction, releasing a-ketoglutarate and ammonium; in doing so, however, it usually uses NAD andproduces NADH. The ammonium release reaction is a key step in the catabolism ofmany amino acids.Glutamate dehydrogenase has a high Km for ammonium. Because ammonium istoxic to animals, ammonium concentration is normally maintained at too low a levelto allow glutamate dehydrogenase