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

Certain types of bacteria can use nitrogen fixation reactions to convert atmospheric N 2 into NH 4 +. 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. The urea cycle uses nitrogen from NH 4 + and the amino acid aspartate to generate urea which is excreted to maintain daily nitrogen balance. Key Concepts in Amino Acid Metabolism

Amino Acid Metabolism The carbon skeleton of amino acids can be harvested for energy converting reactions, whereas, the nitrogen is safely removed to avoid ammonia toxicity.

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 (NH 4 + ) and nitrogen oxides (NO 2 - and NO 3 - ) 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: N H+ 8 e ATP + 16 H 2 O ----> 2 NH 3 + H ADP + 16 P i Nitrogen assimilation using glutamine synthetase and glutamate synthase:  -ketoglutarate + NH ATP + NADPH + H + --> Glutamate + ADP + P i + NADP +

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 N 2 gas into 2 NH 3. Glutamine synthetase - is found in all organisms and it incorporates NH 4 + 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, NH 4 +, and  -ketoglutarate in a redox reaction utilizing either NAD(P) + /NAD(P)H.

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. 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 corn and wheat plants.

Nitrogen fixation Rhizobium bacterium The Haber process Tucson lightning

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.

Nitrogenase reaction is a series of reductions N H+ 8 e ATP + 16 H 2 O ----> 2 NH 3 + H ADP + 16 P i

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.

Nitrogen assimilation in plants Nitrogen assimilation proceeds in one of two ways. First, if NH 4 + levels in the soil are high, plants can use the glutamate dehydrogenase reaction to directly incorporate NH 4 + into the amino acid glutamate using  -ketoglutarate as the carbon skeleton. Note that most often, this reaction releases NH 4 + from glutamate in other contexts.

Nitrogen assimilation in plants A second, and more common way that plants and bacteria incorporate NH 4 + into metabolites, is through a two reaction mechanism that functions when NH 4 + concentrations are low. In this mechanism, the enzyme glutamine synthetase uses ATP in a coupled reaction to form glutamine from glutamate using NH 4 +.

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

The net reaction is nitrogen assimilation

Importantly, the newly acquired nitrogen in glutamate and glutamine is used to synthesize a variety of other amino acids through aminotransferase enzymes such as aspartate aminotransferase.

The Nitrogen Cycle on Planet Earth The Nitrogen Cycle maintains nitrogen balance in our biosphere. The NH 4 + in the soil derived from decomposition, free-living soil bacteria, and man-made fertilizers, is converted to NO 2 - (nitrite) and NO 3 - (nitrate) by soil bacteria that carry out the process of nitrification (e.g., Nitrosomonas and Nitrobacter). Plant roots absorb NO 2 - and NO 3 - present in the soil and convert them back into NH 4 + using nitrite and nitrate reductase enzymes.

Protein and amino acid degradation 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.

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.

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 that cleaves numerous other pancreatic zymogens. Trypsin autoactivates

Protein and amino acid degradation Most eukaryotic cellular proteins are degraded by one of two pathways: 1)ATP-independent process that degrades proteins inside cellular vesicles called lysosomes. 2)ATP-dependent pathway that targets specific proteins for degradation in proteasomes if they contain a polymer of ubiquitin protein covalently attached to lysine residues.

E1 is ubiquinated E1 passes it to E2 E2 + E3 ubiquinate the target protein Ubiquitin is the molecular signal for degradation Ubiquitin is recycled and the protein is degraded

The 26S proteasome consists of a 20S protealytic core and two 19S regulatory complexes that serve as caps to regulate protein entry. The "garbage disposer" of the cell

Amino acids are stripped of their carbon

The Urea Cycle

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? NH HCO aspartate + 3 ATP ---> urea + fumarate + 2 ADP + 2 Pi + AMP + PP i 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.

Glutamate is imported into the mitochondrial matrix where it is metabolized by the enzyme glutamate dehydrogenase to produce NH 4 + which is used to make the urea cycle precursor carbamoyl phosphate.

Citrate cycle

The Urea Cycle 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 C 4 carbon backbone of aspartate gives rise to fumarate: NH CO 2 + aspartate + 3 ATP ---> urea + fumarate + 2 ADP + AMP + 4 P i

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.”

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.

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.

Glucogenic and ketogenic amino acids