Amino Acid Metabolism

How does nitrogenase convert atmospheric N₂ into ammonia?

• Biological nitrogen fixation is catalyzed by the enzyme nitrogenase, contained in microorganisms that live in soil and aquatic environments. Rhizobia produce NH₄⁺, which the plant uses to generate glutamate and glutamine. Animals obtain nitrogen for amino acid synthesis and other biomolecules by eating plants and other animals.

• Industrial nitrogen fixation uses N₂ and H₂ gases under conditions of extreme temperature and pressure to produce NH₃ by the Haber process. Liquid ammonia generated by this industrial process is used to produce agricultural fertilizers, which are the major source of biological nitrogen in developed countries.

• Atmospheric nitrogen fixation occurs when energy from lightning combines N₂ with O₂ to form nitrogen oxides, which are dissolved in rain and fall to Earth. Nitrates produced by lightning are incorporated into plants that convert nitrates and nitrites into NH₃, which is used to synthesize glutamate and glutamine.

• Nitrogenase is a large protein complex in nitrogen-fixing bacteria that catalyzes an ATP-dependent redox reaction, converting N₂ into 2 NH₃. Three rounds of nitrogen reduction sequentially convert N₂ → diimine → hydrazine → 2 NH₃; each reduction transfers 1 e⁻ to the nitrogenase complex with the hydrolysis of 2 ATP. Because 6 e⁻ are required to generate 2 NH₃ from the reduction of N₂, and H₂ is produced in a wasteful side reaction, a total of 8 e⁻ and 16 ATP are required.

• The nitrogenase reaction is inhibited by O₂, so microorganisms have evolved mechanisms to limit O₂ access to the enzyme active site: (1) Klebsiella pneumoniae synthesizes the protein components of the nitrogenase complex only when it is living in an anaerobic environment. (2) Azotobacter vinelandii decreases local O₂ concentrations by increasing flux through the electron transport system to rapidly reduce O₂ to H₂O. (3) Sinorhizobium meliloti invades the roots of leguminous plants, which synthesize a heme-containing protein called leghemoglobin that sequesters O₂ away from the nitrogenase complex.

• A major source of nitrogen in the soil is NH₄⁺, which is produced by the degradation of plant and animal parts by invertebrates, bacteria, and fungi that live in the soil. Another major source of nitrogen in the soil comes from the process of nitrification, which bacteria use to convert NH₄⁺ in the soil into NO₂⁻ (nitrite), and then into NO₃⁻ (nitrate).

• The nitrogen cycle refers to the recycling of soil NH₄⁺, NO₂⁻, and NO₃⁻ back to either plant roots or, through a process of bacterial denitrification, to atmospheric N₂.

How is ammonia assimilated into glutamate and glutamine?

• Plants and bacteria use the available NH₄⁺ generated by nitrogen fixation to synthesize the amino acids glutamate and glutamine. Glutamate is the source of nitrogen in amino acid biosynthesis through the action of aminotransferase enzymes. Glutamine is the primary source of amino groups for the biosynthesis of nucleotide bases and carbamoyl phosphate, a metabolite in the urea cycle.

• The incorporation of NH₄⁺ into glutamate and glutamine is called ammonia assimilation and is mediated by three enzymes: (1) glutamine synthetase, (2) glutamate synthase, and (3) glutamate dehydrogenase.

• The combined action of glutamine synthetase and glutamate synthase catalyzes a net reaction that generates glutamate from α-ketoglutarate and NH₄⁺. Glutamate dehydrogenase catalyzes the same reaction, but only under conditions of very high NH₄⁺ levels, such as those after application of agricultural fertilizers in fields (ΔG°′ = +30 kJ/mol).

• Glutamine synthetase is regulated by both feedback inhibition and covalent attachment of AMP to a tyrosine residue in the enzyme. Adenylylation of glutamine synthetase increases its sensitivity to allosteric inhibitors, which include the metabolites carbamoyl phosphate, AMP, CTP, histidine, tryptophan, and serine.

• Aminotransferases play an important role in amino acid degradation and synthesis by transferring amino groups between amino acids and α-keto acids. For example, aspartate aminotransferase transfers the α amino group of aspartate to α-ketoglutarate to form glutamate and oxaloacetate.

How are proteins targeted for degradation by the ubiquitin–proteasome system?

• Nitrogen cannot be stored in the body 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, the daily intake of nitrogen equals the amount of nitrogen lost by excretion.

• Plants and bacteria synthesize all 20 amino acids; however, animals depend on protein in their diets to obtain the 10 essential amino acids they require for growth and development. Protein digestion in humans occurs in the stomach and small intestine, where proteases cleave proteins to yield amino acids and oligopeptides.

• The interior of lysosomal vesicles are acidic, providing optimal conditions for protein unfolding and proteolytic degradation. Proteasomes degrade ubiquitinated proteins by using three distinct protease activities located within the internal chamber of the catalytic core.

• Proteins destined for proteasomal degradation are tagged on lysine residues by covalent linkage of ubiquitin through its carboxyl-terminal glycine residue. The proteasome consists of a 20S core particle and two 19S regulatory particles; the 19S complexes serve as caps to regulate protein entry into and exit from the 20S proteolytic core.

• Ubiquitinating enzymes recognize specific residues at the N terminus of the target protein or a structural property such as misfolding. There are three classes of ubiquitinating enzymes: E1 enzymes attach ubiquitin to E2 enzymes, E2 enzymes conjugate ubiquitin to target proteins, and E3 enzymes recognize target proteins and facilitate ubiquitination by interacting directly with E2–ubiquitin and the target protein.

• Ubiquitin activation is ATP dependent and links the terminal carboxyl group of Gly76 in ubiquitin to a Cys residue in the E1 enzyme. Transfer of this ubiquitin to a Cys residue in E2 releases E1 and leads to the formation of an E2–ubiquitin–E3 complex. Ubiquitination of a Lys residue on target proteins initiates polyubiquitination, which links at least four ubiquitin subunits together through Gly76–Lys48 linkages.

• Target proteins having a Phe, Leu, Asp, Lys, or Arg residue at the N terminus are ubiquitinated by E2–ubiquitin–E3 complexes that follow the “N-end rule” of protein degradation. The N-end rule refers to the propensity of polypeptides with specific N-terminal amino acids to have short half-lives in cells.

How does the urea cycle convert excess nitrogen into urea?

• Glutamate and glutamine function as the primary nitrogen carriers in most organisms. Mammals excrete excess nitrogen as urea, which is synthesized in the liver. Fish and aquatic amphibians excrete NH₄⁺ directly into water, whereas terrestrial amphibians excrete nitrogen as urea or uric acid.

• The two nitrogens in urea are derived from (1) NH₄⁺ released from the deamination of glutamate and glutamine and (2) incorporation of aspartate into the urea cycle intermediate argininosuccinate. The carbon atom in urea comes from CO₂ (HCO₃⁻) produced in the citrate cycle, and the oxygen atom is derived from H₂O.

• In the first reaction of the urea cycle, HCO₃⁻ and NH₄⁺ are used to synthesize carbamoyl phosphate, which is then combined with ornithine to form citrulline. Citrulline is exported to the cytosol and activated by AMP before being converted to argininosuccinate when aspartate displaces the AMP. Argininosuccinate is cleaved to yield fumarate and arginine, followed by arginase cleavage to produce urea.

• The urea cycle and citrate cycle are metabolically linked through the shared intermediate fumarate, which provides the carbon backbone for aspartate by supplying the oxaloacetate needed in the aspartate aminotransferase reaction. These reactions are called the “Krebs bicycle,” or aspartate–argininosuccinate shunt.

• Urea cycle enzyme deficiencies result in hyperammonemia and a buildup of glutamine and glutamate, which function as osmolytes that can cause brain swelling and associated neurologic symptoms. Some urea cycle disorders can be treated by restricting dietary protein or by providing an alternative path for nitrogen excretion.

How are the carbon skeletons of amino acids degraded into glucogenic and ketogenic products?

• The carbon backbones of 11 amino acids can be converted into pyruvate or acetyl-CoA, which are used for energy conversion by the citrate cycle and oxidative phosphorylation reactions. The other nine amino acids are converted to α-ketoglutarate, fumarate, succinyl-CoA, and oxaloacetate for glucose synthesis.

• Amino acids that give rise to pyruvate or citrate cycle intermediates are called glucogenic because pyruvate and oxaloacetate are precursors in the gluconeogenic pathway. In contrast, amino acids converted into acetyl-CoA or acetoacetyl-CoA are called ketogenic amino acids because they can give rise to ketone bodies.

• Three interconnected amino acid degradation pathways cover 13 amino acids: (1) amino acids that directly (glycine, serine, alanine, and cysteine) or indirectly (threonine and tryptophan) give rise to pyruvate; (2) amino acids that generate α-ketoglutarate (arginine, histidine, proline, glutamate, and glutamine); and (3) the degradation of phenylalanine to tyrosine.

• Tetrahydrofolate (THF) is a coenzyme that functions as a one-carbon (C₁) donor in a number of metabolic reactions and consists of the amino acid glutamate attached to p-aminobenzoate and 6-methylpterin. The five C₁ transfer groups of THF derivatives are (1) a methyl group (—CH₃), (2) a methylene group (—CH₂—), (3) a formyl group (—CHO), (4) a formimino group (—CH=NH), and (5) a methenyl group (—CH=).

• Defects in the gene encoding homogentisate-1,2-dioxygenase, an enzyme in the phenylalanine and tyrosine degradation pathways, cause the disease alkaptonuria, or black urine disease. Large amounts of homogentisate accumulate in the urine and cause the black color upon oxidation.

• Phenylketonuria is due to defects in the enzyme phenylalanine hydroxylase, which converts phenylalanine to tyrosine. Phenylketonuria symptoms are caused by the accumulation of phenylalanine metabolites (phenylpyruvate, phenylacetate, and phenyllactate). The primary treatment for phenylketonuria is to limit phenylalanine in the diet, beginning shortly after birth. This includes avoiding foods containing the artificial sweetener aspartame.

What are the metabolic precursors of amino acid biosynthesis?

• The side chains of amino acids are derived from seven metabolic intermediates in three metabolic pathways: (1) the glycolytic pathway (3-phosphoglycerate, phosphoenolpyruvate, and pyruvate); (2) the pentose phosphate pathway (ribose-5-phosphate and erythrose-4-phosphate); and (3) the citrate cycle (α-ketoglutarate and oxaloacetate).

• The structures of the essential amino acids are more complex than those of the nonessential amino acids; alanine, serine, and aspartate are synthesized by all organisms in pathways requiring only a few enzymes, whereas plants and bacteria synthesize tryptophan, histidine, and methionine by pathways requiring a large number of enzymes.

• Regulation of metabolic flux through amino acid biosynthetic pathways is tightly controlled to maintain a pool of amino acids that optimally supports protein synthesis; feedback inhibition is key to modulating flux through linked amino acid biosynthetic pathways.

• Biosynthesis of three nonessential amino acids (alanine, aspartate, and asparagine) and six essential amino acids (methionine, threonine, lysine, isoleucine, valine, and leucine) involves two interconnected pathways in bacteria that use pyruvate and oxaloacetate as precursors.

What is the molecular basis for glyphosate (Roundup) resistance?

• Aromatic amino acids are synthesized in plants, fungi, and bacteria by the shikimate pathway, which uses the substrates phosphoenolpyruvate and erythrose-4-phosphate to generate chorismate, the metabolic precursor to tryptophan, tyrosine, and phenylalanine.

• Animal cells do not contain the enzymes required for chorismate biosynthesis. Glyphosate (Roundup) inhibits the enzyme EPSP synthase, which is required to convert shikimate-3-phosphate to 5-enolpyruvylshikimate-3-phosphate (EPSP). Glyphosate-treated plants die because they are deficient in tryptophan, tyrosine, and phenylalanine.

• Roundup Ready soybeans contain a bacterial gene coding for the enzyme CP4 EPSP synthase, which does not bind glyphosate and thereby provides glyphosate resistance to the plant. Farmers growing Roundup Ready soybeans spray their crops with glyphosate to kill weeds that compete with the crop plants for nutrients and water.

What are functions of some of the most common amino acid derivatives?

• The iron porphyrin ring of hemoglobin, myoglobin, and the cytochromes is derived from the amino acid glycine and is synthesized by a complex pathway requiring eight enzymes localized to either the mitochondrial matrix or the cytosol.

• Heme biosynthesis takes place in erythrocyte precursors in the bone marrow to produce hemoglobin and in liver cells to provide heme for enzymes. Porphyrias result from defects in enzymes in the heme biosynthetic pathway; acute intermittent porphyria is due to a dominant mutation in the porphobilinogen deaminase gene.

• One of the products of hemoglobin degradation is bilirubin, a yellow metabolite that can accumulate in the blood and cause a condition called jaundice. Most bilirubin degradation products produced each day are excreted in the feces and urine.

• Tyrosine is the precursor to biomolecules required in metabolic signaling and neurotransmission (epinephrine and dopamine) and to pigments (eumelanins and pheomelanins).

• Two human diseases related to tyrosine metabolism are Parkinson disease, which is caused by a loss of dopamine-producing cells in the brain, and albinism, which results from genetic defects in the enzyme tyrosinase.

• Nitric oxide (NO) is a soluble gas that functions as a potent vasodilator. NO is produced from arginine in a two-step oxidation reaction catalyzed by the enzyme nitric oxide synthase. Humans have three nitric oxide synthase enzymes: (1) the endothelial form involved in vasodilation, (2) the neuronal form required for neuronal signaling in the brain, and (3) the inducible form that is present in immune cells.

• NO-mediated vasodilation in smooth muscle cells is induced by acetylcholine activation of calcium signaling through the muscarinic acetylcholine receptor in endothelial cells, which stimulates calmodulin-dependent endothelial nitric oxide synthase activity. Diffusion of NO into nearby smooth muscle cells activates soluble guanylate cyclase and production of cGMP, which binds to protein kinase G, leading to phosphorylation of target proteins.