Introduction to amino acid metabolism Overview The body has a small pool of free amino acids The pool is dynamic and is constantly being used as a source of substrate for various reactions and is constantly being replenished Free amino acids are not stored except as part of larger molecules i e proteins Amino acids can be used for a variety of functions The primary function of amino acids is to act as the monomer unit in protein synthesis Amino acids can be also used as substrates for biosynthetic reactions the nucleotide bases heme and a number of hormones and neurotransmitters are derived from amino acids Finally the carbon skeleton of all of the amino acids broken down for energy Nitrogen metabolism Unlike glucose or fatty acids amino acids contain nitrogen Biologically relevant inorganic nitrogen molecules include dinitrogen N2 nitrogen oxides including NO2 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 for this toxicity are incompletely understood but most symptoms involve altered brain function suggesting that the central nervous system is vulnerable to ammoniuminduced damage Ammonium therefore must be handled carefully and physiological nitrogen metabolism must take this into account 4 Ammonium is the protonated form of ammonia NH3 ammonium is the major species in aqueous solution Copyright 2000 2003 Mark Brandt Ph D 24 Ammonium can come from several sources depending on the organism 1 organic nitrogen nitrogen attached to organic molecules that can be metabolized 2 free ammonium 3 nitrogen oxides especially nitrate and 4 dinitrogen Nitrogen fixation N2 is inaccessible to most organisms because of the strong bond between the nitrogen atoms Although the conversion of N2 to NH3 has a G of about 33 kJ 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 called nitrogen 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 nif genes which code for nitrogenase and its accessory proteins Nitrogenase requires iron sulfur and molybdenum as cofactors Nitrogenase is rapidly denatured by oxygen and therefore requires an oxygen free environment Legumes have leghemoglobin a monomeric globin with high oxygen affinity It functions to protect the bacteria from free oxygen by transferring oxygen only to the bacterial cytochrome c oxidase Free living bacteria either live in anaerobic environments or use uncoupling agents to increase their rate of oxygen reduction to protect their nitrogenase complex The nitrogen fixation reaction is expensive at least 16 ATP are required to overcome the energy barrier in dinitrogen The actual energy requirements are usually higher than the minimum stoichiometry shown below due to wasteful side reactions This means that organisms capable of fixing nitrogen have considerable energy requirements Legumes use 20 of their ATP production to supply energy for their symbiotic bacteria The nitrogen fixation process requires electrons In free living cyanobacteria the electrons are derived from a photosynthetic electron transport chain The symbiotic nitrogen fixing bacteria of legumes are present in root nodules because they are not exposed to sunlight these bacteria must use electrons from metabolic sources such as the pyruvate dehydrogenase reaction to drive nitrogen reduction Humans can only perform N2 reduction using technological assistance The Haber process invented shortly before World War I uses high pressures of hydrogen gas 200 atmospheres and temperatures 700 K to achieve what the bacteria manage at ambient temperature and pressure Copyright 2000 2003 Mark Brandt Ph D 25 Nitrogen assimilation Plants can use either ammonium or nitrogen oxides especially nitrate as sources of usable nitrogen Nitrate is formed by microorganisms that can use ammonium as an energy 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 why fertilizer can be dangerous The explosive potential of the common fertilizer NH4NO3 when combined with readily available carbon compounds such as diesel oil has led to some limits on the sale of this material Nitrate reduction requires electrons derived from photosynthesis to produce ammonium The reduction of nitrate must be followed by ammonium fixation the process of attaching ammonium ions to carbon compounds The reactions used for this purpose are discussed below Unlike plants animals use organic nitrogen derived from their diet for essentially all of their nitrogen requirements Animals require nitrogen in reduced form and release most nitrogen in reduced form in general animals cannot reduce nitrogen oxides and do not excrete these compounds Most organisms have three major reactions that incorporate inorganic nitrogen into organic compounds These reactions are catalyzed by glutamate dehydrogenase glutamine synthetase and one isozyme of carbamoyl phosphate synthase this last enzyme we will discuss later during the discussion of the urea cycle In addition one pathway for glycine synthesis uses inorganic ammonium under most 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 bind ammonium to a ketoglutarate It can also catalyze the reverse reaction releasing aketoglutarate and ammonium in doing so however it usually uses NAD and produces NADH The ammonium release reaction is a key step in the catabolism of many amino acids Glutamate dehydrogenase has a high Km for ammonium Because ammonium is toxic to animals ammonium concentration is normally maintained at too low a level to allow glutamate dehydrogenase to synthesize significant amounts of glutamate Instead another reaction catalyzed by glutamine synthetase is more important for ammonium fixation in most species In many plants glutamine synthetase is the sole ammonium fixation enzyme Glutamine synthetase uses ATP as the source of energy for the reaction Copyright 2000 2003 Mark Brandt Ph D 26 While