Bioc 460 - Dr. Miesfeld Spring 2008 1 of 14 pages Amino Acid Metabolism 1 Key Concepts - Nitrogen fixation and assimilation - Protein and amino acid degradation - The Urea cycle - Glucogenic and ketogenic amino acids Key Concept Questions In Amino Acid Metabolism How is atmospheric nitrogen converted into ammonia as a nitrogen source for plants? Which three amino acids serve as the nitrogen donors in urea synthesis? Nitrogen fixation and assimilation by plants and bacteria After carbon, nitrogen is the second most abundant element in the biosphere, and in addition to its presence in amino acids and nucleotides, it is also found in some carbohydrates (glucosamine) and lipids (sphingosine), as well as, the enzyme cofactors thiamine, NAD+, and FAD. Nitrogen in biological compounds ultimately comes from nitrogen gas (N2) which constitutes 80% of our atmosphere. However, N2 must first be reduced to NH3 (the ionized form of ammonia in solution is NH4+) by the process of nitrogen fixation, or oxidized to nitrate (NO3-) by atmospheric lightning, before it can be used by other liver organisms. Nitrogen fixation in nature is carried out by certain types of soil bacteria that live in both the soil and aquatic environments. Rhizobium is an example of a nitrogen-fixing soil bacteria that lives symbiotically with leguminous plants such as beans and alfalfa and has an important role in agriculture by reducing the need for commercial fertilizers. Plants cannot carry out nitrogen fixation on their own, but they can incorporate NH4+ they obtain from the environment into the amino acids glutamate and glutamine through a process called nitrogen assimilation. When animals eat plants, amino acids and nucleotides provide the nitrogen needed to synthesize a variety of biomolecules. Before looking at nitrogen metabolism in a little more detail, we need to answer our four pathway questions about nitrogen fixation and assimilation. 1. What purpose does nitrogen fixation and assimilation serve in the biosphere? • Nitrogen fixation takes place in bacteria and is the primary process by which atmospheric N2 gas is converted to ammonia (NH4+) and nitrogen oxides (NO2- and NO3-) in the biosphere. Two other nitrogen fixation processes are industrial (Haber process) and atmospheric (lightening). • Nitrogen assimilation is the process by which plants and bacteria incorporate NH4+ into organic compounds, most often the NH4+ is incorporated into the amino acids glutamate and glutamine. 2. What are the net reactions of nitrogen fixation and assimilation by plants and bacteria? Nitrogen fixation in bacteria is mediated by the nitrogenase enzyme complex: N2 + 8 H+ 8 e- + 16 ATP + 16 H2O ----> 2 NH3 + H2 + 16 ADP + 16 Pi Nitrogen assimilation in plants using the enzymes glutamine synthetase and glutamate synthase: α-ketoglutarate + NH4+ + ATP + NADPH + H+ --> Glutamate + ADP + Pi + NADP+Bioc 460 - Dr. Miesfeld Spring 2008 2 of 14 pages Figure 1. 3. What are the key enzymes in nitrogen fixation and assimilation in plants and bacteria? Bacterial nitrogenase complex – is the enzyme that uses redox reactions coupled to ATP hydrolysis to convert N2 gas into 2 NH3. The enzyme has two functional components, dinitrogenase reductase that contains the binding site for ATP and 4Fe-4S redox center, and dinitrogenase which carries out the N2 reduction reaction using Fe-Mo and FeS redox centers. Glutamine synthetase - is found in all organisms and it incorporates NH4+ into glutamate to form glutamine through an ATP coupled redox reaction. The activity of glutamine synthetase is regulated by allosteric inhibitors (amino acids, AMP), and by covalent modification which is mediated by adenylation. Glutamate synthase - is found in bacteria, plants, and some insects, and it works in concert with glutamine synthetase to replenish glutamate so that the glutamine synthetase reaction is not substrate limited. Glutamate synthase converts α-ketoglutarate and glutamine to 2 glutamate. Glutamate dehydrogenase - is found in all organisms and it interconverts glutamate, NH4+, and α-ketoglutarate in a redox reaction utilizing either NAD(P)+/NAD(P)H. Under conditions of high NH4+ concentrations in nature, e.g., fertilizer applications in crop fields, glutamate dehydrogenase can assimilate NH4+ into glutamate, however, in animals, glutamate dehydrogenase most often generates NH4+ from glutamate to initiate the process of nitrogen excretion as urea or uric acid. 4. What are examples of nitrogen fixation and assimilation in real life? Natural fertilizers can be used in organic farming to reduce the dependence on industrial sources of nitrogen. The two most common sources of natural fertilizers are manure, if livestock are readily available, and crop rotation practices in which leguminous plants such as soy bean or clover, are planted in alternate seasons with nonleguminous crop plants such as corn and wheat. By plowing under the leguminous plants, the nitrogen contained in the plants is released into the soil and processed by soil bacteria to provide nitrogenous compounds for the corn and wheat plants. Nitrogen fixation In order to obtain nitrogen from the atmosphere for incorporation into biomolecules, the triple bond of N2 must be broken. However, this is not easily done considering that the bond energy of N2 is a staggering 930 kJ/mol. To overcome this high energy barrier, one of three processes are required, 1) biological fixation by bacteria that reduce N2 to NH3 through an ATP-dependent process requiring the multisubunit enzyme complex nitrogenase, 2) industrial fixation by the Haber process in which N2 and H2 gases are heated to ~500ºC under a pressure of ~250 atmospheres (~350 kilopascals) to produce liquid ammonia which is used commercially to make fertilizer (figure 1), or 3) atmospheric fixation as a result of lightning which breaks the N2 triple bond and allows nitrogen to combine with oxygen to form nitrogen oxides that are dissolved in rain and fall to earth. It is estimated that ~90% of the nitrogen incorporated our biosphere comes from biological and industrial fixation, of which almost half of this is ammonia contained in agricultural fertilizers.Bioc 460 - Dr. Miesfeld Spring 2008 3 of 14 pages Figure 2. Figure 3. Fritz Haber, a German chemist, received the 1918 Nobel Prize in Chemistry for his development of industrial ammonia synthesis which revolutionized
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