Bioc 460 Spring 2008 - Lecture 38 (Miesfeld)

1. Amino Acid Metabolism 1:Nitrogen fixation and assimilation, amino acid degradation, the urea cycle Bioc 460 Spring 2008 - Lecture 38 (Miesfeld) Glutamine synthetase converts glutamate to glutamine through a nitrogen assimilation reaction Red clover is a leguminous plant that is often used in crop rotation strategies Urea is a nitrogen-containing metabolite that efficiently removes toxic ammonia

2. Key Concepts in Amino Acid Metabolism • Certain types of bacteria can use nitrogen fixation reactions to convert atmospheric N2 into NH4+which can then be incorporated into the amino acids glutamate and glutamine by plants. • The enzymes glutamate synthase, glutamine synthetase, glutamate dehydrogenase, and aminotransferases are responsible for the vast majority of nitrogen metabolizing reactions in most organisms. • Protein degradation by the proteasomal complex releases oligopeptides that are degraded into individual amino acids that are either recycled for protein synthesis, or deaminated to salvage reduced carbon for energy. • The urea cycle uses nitrogen from NH4+ and the amino acid aspartate to generate urea which is excreted to maintain daily nitrogen balance.

3. Amino Acid Metabolism Digestion of dietary protein, and protein turnover within cells, especially muscle cells, provides amino acids that serve as metabolic intermediates in anabolic reactions requiring nitrogen-containing precursors. The carbon skeleton of amino acids can be harvested for energy converting reactions, whereas, the nitrogen is safely removed to avoid ammonia toxicity.

4. Nitrogen fixation and assimilation by plants and bacteria Nitrogen is the second most abundant element in the biosphere after carbon, 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. 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.

5. Nitrogen fixation and assimilation by plants and bacteria 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. Nitrogen assimilation incorporates this ammonia into amino acids, primarily glutamate and glutamine. 2. What are the net reactions of nitrogen fixation and assimilation by plants and bacteria? Nitrogen fixation 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 using glutamine synthetase and glutamate synthase: a-ketoglutarate + NH4+ + ATP + NADPH + H+ --> Glutamate + ADP + Pi + NADP+

6. Nitrogen fixation and assimilation by plants and bacteria 3. What are the key enzymes in nitrogen fixation and assimilation? Bacterial nitrogenase complex – is the enzyme that uses redox reactions coupled to ATP hydrolysis to convert N2 gas into 2 NH3. Glutamine synthetase - is found in all organisms and it incorporates NH4+ into glutamate to form glutamine through an ATP coupled redox reaction. 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 dehydrogenase - is found in all organisms and it interconverts glutamate, NH4+, and a-ketoglutarate in a redox reaction utilizing either NAD(P)+/NAD(P)H.

7. Nitrogen fixation and assimilation by plants and bacteria 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.

8. 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 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. 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.

9. Nitrogen fixation Rhizobium bacterium Tucson lightning The Haber process

10. Nitrogen fixation in bacteria Biological nitrogen fixation by bacteria requires the activity of nitrogenase, a large protein complex consisting of two functional components. One component is called dinitrogenase reductase (Fe-protein) which consists of two identical subunits that each contain a binding site for ATP, and a single 4Fe-4S redox center liganded to cysteine residues in the two subunits. The second component of the complex is called dinitrogenase (MoFe-protein) and it catalyzes the reduction of N2 to generate 2 NH3.

11. Nitrogen fixation in bacteria • The nitrogenase reaction requires 16 ATP to overcome the large energy barrier in the N2 triple bond. • N2 + 8 H+ 8 e- + 16 ATP + 16 H2O ----> 2 NH3 + H2 + 16 ADP + 16 Pi The reduction of N2 to 2 NH3 takes place in the Mo-Fe reaction center of the dinitrogenase component and requires three discrete steps that each utilize 2 H+ and 2 e-.

12. Nitrogen fixation in bacteria Rhizobium meiloti is one of the bacterial species that is capable of nitrogen fixation. This bacterial species invades the roots of leguminous plants through tubular structures called infection threads. Once in side the plant cell, the bacterium loses its cell wall and becomes a bacteroid, containing an inner and outer membrane. The plant provides citrate cycle intermediates such as fumarate and malate to the bacteroids which use them as oxidizable energy sources to generate NADH. In turn, the nitrogenase complex of the bacteroid generates NH3 which is used to synthesize amino acids such as glutamate and aspartate that the plant can use as source of nitrogen.

13. Nitrogen assimilation in plants Plants and bacteria, but not animals, use nitrogen assimilation reactions to synthesize nitrogen-containing metabolites, primarily amino acids. Nitrogen assimilation proceeds in one of two ways. First, if NH4+ levels in the soil are high, plants can use the glutamate dehydrogenase reaction to directly incorporate NH4+ into the amino acid glutamate using a-ketoglutarate as the carbon skeleton. Animals also contain glutamate dehydrogenase, but because of low levels of free NH4+ in cells (NH4+ is toxic), and correspondingly high levels of glutamate, the reaction is run in the reverse direction as a way to deaminate glutamate and form nitrogenous waste products such as urea and uric acid.

14. Nitrogen assimilation in plants A second, and more common way that plants and bacteria incorporate NH4+ into metabolites, is through a two reaction mechanism that functions when NH4+ concentrations are low. In this mechanism, the enzyme glutamine synthetase uses ATP in a coupled reaction to form glutamine from glutamate using NH4+.

15. Nitrogen assimilation in plants Next, the glutamine is combined with a-ketoglutarate in a reaction catalyzed by the enzyme glutamate synthase to form two molecules of glutamate (glutamine contains two nitrogens).

16. Nitrogen assimilation in plants

17. Nitrogen assimilation in plants Importantly, the newly acquired nitrogen in glutamate and glutamine is used to synthesize a variety of other amino acids through aminotransferase enzymes that convert glutamate and a-keto acids into a-ketoglutarate and the corresponding a-amino acid.

18. The Nitrogen Cycle on Planet Earth The Nitrogen Cycle maintains nitrogen balance in our biosphere. Atmospheric nitrogen is converted to NH3 by biological, industrial and atmospheric fixation processes. Bacteria in the soil convert nitrogen to NH3 either as symbionts with leguminous plants (e.g., Rhizobium), or as free-living organisms (e.g., Azotobacter). The NH4+ in the soil derived from decomposition, free-living soil bacteria, and man-made fertilizers, is converted to NO2- (nitrite) and NO3- (nitrate) by soil bacteria that carry out the process of nitrification (e.g., Nitrosomonas and Nitrobacter). Plant roots absorb NO2- and NO3- present in the soil and convert them back into NH4+ using nitrite and nitrate reductase enzymes.

20. Protein and amino acid degradation Unlike glucose which can be stored in the body as glycogen, or converted to acetyl CoA and stored as fatty acids, nitrogen cannot be stored in a useable form because NH4+ is toxic. Therefore, nitrogen lost as a result of protein and nucleic acid degradation, must be replaced from the diet. When an individual is in nitrogen balance, it means that their daily intake of nitrogen, primarily in the form of protein, equals the amount of nitrogen lost by excretion in the feces and urine. A normal healthy adult needs about 400 grams of protein per day to maintain nitrogen balance. In contrast, young children and pregnant women have a positive nitrogen balance because they accumulate nitrogen in the form of new protein which is needed to support tissue growth. Negative nitrogen balance is a sign of disease or starvation and occurs in individuals with elevated rates of protein breakdown (loss of muscle tissue) or an inability to obtain sufficient amounts of amino acids in their diet.

21. Protein and amino acid degradation Plants and bacteria have the necessary enzymes to synthesize all 20 amino acids, however, animals depend on protein in their diets to obtain the ten essential amino acids they require for growth and development. Protein digestion in humans takes place in the stomach and the small intestine where proteases cleave the peptide bond to yield amino acids and small oligopeptides.

22. Protein and amino acid degradation The duodenum secretes enteropeptidase, a protease that specifically activates several protealytic zymogens released from the pancreas. One of these proteases is the pancreatic zymogen trypsinogen which is cleaved to form the endopeptidase trypsin which cleaves numerous pancreatic zymogens, including chymotrypsinogen, proelastase and procarboxypepetidases A and B, as well as, trypsinogen itself to amplify the protealytic cascade.

23. Protein and amino acid degradation Another source of free amino acids in the body comes from degradation of cellular proteins which occurs continuously in all cells. Most eukaryotic cellular proteins are degraded by one of two pathways: an ATP-independent process that degrades proteins inside cellular vesicles called lysosomes, and an ATP-dependent pathway that targets specific proteins for degradation in proteasomes if they contain a polymer of ubiquitin protein covalently attached to lysine residues. In order for proteins to be degraded by the proteasome, they are first "tagged" on lysine residues by covalent linkage of ubiquitin. Ubiquitin is a 76 amino acid protein found in all eukaryotic cells that is specifically attached to proteins by ubiquitin ligating enzymes.

25. The proteasome consists of a 20S protealytic core, so named because of its sedimentation properties in a density gradient (S is Svedberg units), and two 19S regulatory complexes that serve as caps to regulate protein entry into the protealytic core. The intact proteasome is 26S and has often been called the "garbage disposer" of the cell.

26. Amino acids are stripped of their carbon

27. The Urea Cycle Glutamate and glutamine function as the primary nitrogen carriers in most organisms. In mammals, this nitrogen ends up in the liver where it is converted to urea. The two nitrogens in urea are derived from the NH4+ released when glutamate or glutamine are deaminated, and from aspartate which is formed when oxaloacetate is transaminated by aspartate aminotransferase. The carbon atom in urea comes from CO2 (HCO3-) that is produced in the mitochondrial matrix by the citrate cycle (the oxygen atom is derived from H2O in the final reaction of the cycle).

28. The Urea Cycle

29. 1. What does the urea cycle accomplish for the organism? • Urea synthesis provides an efficient mechanism for land animals to remove excess nitrogen from the body. Urea is synthesized in the liver and exported to the kidneys where it enters the bladder. • 2. What is the net reaction of the urea cycle? • NH4+ + HCO3- + aspartate + 3 ATP ---> • urea + fumarate + 2 ADP + 2 Pi + AMP + PPi • 3. What is the key regulated enzyme in urea synthesis? • Carbamoyl phosphate synthetase I – catalyzes the commitment step in the urea cycle; the activity of this mitochondrial enzyme is activated by N-acetylglutamate in response to elevated levels of glutamate and arginine. • 4. What is an example of the urea cycle in real life? • Argininosuccinase deficiency inhibits flux through the urea cycle and causes hyperammonemia and neurological symptoms. This metabolic disease can be treated with a low protein diet that is supplemented with arginine, thereby resulting in argininosuccinate excretion a substitute for urea.

30. The Urea Cycle Amino acids are transported to the liver where the nitrogen is removed and used for urea synthesis. There are three sources of these amino acids: amino acids derived from the digestion of dietary proteins the amino acid glutamine, which is generated from glutamate and NH4+ in peripheral tissues by glutamine synthetase the amino acid alanine, which is formed by the alanine aminotransferase reaction as a way to remove excess nitrogen from exercising (or starving) skeletal muscle.

31. Glutamate is imported into the mitochondrial matrix where it is metabolized by the enzyme glutamate dehydrogenase to produce NH4+ which is used to make the urea cycle precursor carbamoyl phosphate.

33. The Urea Cycle In the last step of the urea cycle, the enzyme arginase converts arginine to urea and ornithine to complete the cycle. Ornithine has the same role in the urea cycle as oxaloacetate does in the citrate cycle, namely, as both the product of the last reaction and the substrate of the first reaction. By including the pyrophosphatase reaction (argininosuccinate synthetase reaction), it can be seen that four high energy phosphate bonds are required (4 ATP equivalents) for every molecule of urea that is synthesized, and moreover, that the C4 carbon backbone of aspartate gives rise to fumarate: NH4+ + CO2 + aspartate + 3 ATP ---> urea + fumarate + 2 ADP + AMP + 4 Pi

34. The aspartate-argininosuccinate shunt converts fumarate, produced in the cytosol by the urea cycle, into malate that is used to make oxaloacetate in the citrate cycle, thereby, forming the “Krebs bicycle.”

35. Enzyme Deficiencies in the Urea Cycle While complete loss of a urea cycle enzyme causes death shortly after birth, deficiencies in urea cycle enzymes results in hyperammonemia (elevated ammonia levels in the blood). Most urea cycle disorders also lead to a build-up of glutamine and glutamate which function as osmolites that can cause brain swelling and associated neurological symptoms. Fortunately, it is possible to treat some of the urea cycle disorders by restricting dietary protein as a means to limit nitrogen intake. For example, argininosuccinase deficiency can be treated effectively by putting patients on a protein-depleted diet that is supplemented with high doses of L-arginine. This regimen increases flux through a "short-circuited" urea cycle by converting arginine to ornithine, which combines with carbamoyl phosphate and aspartate to generate argininosuccinate.

36. Enzyme Deficiencies in the Urea Cycle Since argininosuccinate is soluble and can be excreted in the urine, it functions as a metabolic replacement for urea. Supplementing the diet with ornithine would give the same result, but it is more feasible to use arginine.

37. Degradation of glucogenic and ketogenic amino acids The carbon backbones of eleven of the twenty standard amino acids can be converted into pyruvate or acetyl-CoA, which can then be used for energy conversion by the citrate cycle and oxidative phosphorylation reactions. The other nine amino acids are converted to the citrate cycle intermediates -ketoglutarate, fumarate, succinyl-CoA, and oxaloacetate, which can be used for glucose synthesis by conversion of oxaloacetate to phosphoenolypyruvate. Under normal conditions, amino acid degradation accounts for ~10-15% of the metabolic fuel for animals, more so for animals with high protein diets or during starvation when muscle protein is degraded.