8UreaCycle - 8. The urea cycle and amino acid catabolism...

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Unformatted text preview: 8. The urea cycle and amino acid catabolism and biosynthesis 1 amino acids Most mammals convert amino-acid nitrogen to urea for excretion most terrestrial vertebrates The carbon chains are broken down to molecules that feed into the TCA cycle. NH4+ Some animals excrete NH4+ or uric acid. fish & other aquatic vertebrates birds & reptiles O O H2N-C-NH2 urea NH4+ ammonium ion HN uric acid O H N O N H N H 2 The urea cycle HCO3- 2 ATP NH4+ ornithine 2 ADP + Pi carbamoyl OO H2N-C-O-P-O- phosphate OPi NH3+ H2N-CH2CH2CH2CH-CO2- O H2N-C-NH2 urea urea H2O arginine 3 cytosol mitochondria citrulline NH3+ O H2N-C-NH-CH2CH2CH2CH-CO2- CO2- Asp O2C-CH2CH-NH3+ ATP AMP + PPi CO2NH2+ NH3+ O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2- NH3+ NH2+ H2N-C-NH-CH2CH2CH2CH-CO2- argininosuccinate O2C-CH=CH-CO2- - fumarate Incorporation of ammonia into urea begins with formation of carbamoyl phosphate OO H2N-C-O-P-OO2 ADP + Pi NH4+ + HCO32 ATP carbamoyl phosphate This occurs in the mitochondrial matrix. Carbamoyl-phosphate synthetase-I catalyzes the reaction in three steps, using two molecules of ATP: HCO3ATP (1) ADP OO HO-C-O-P-OONH4+ (2) carbamate O H2N-C-O- Pi carbonicphosphoric acid anhydride ATP ADP (3) OO H2N-C-O-P-OO- 4 Carbamoyl phosphate reacts with ornithine to form citrulline OO H2N-C-O-P-OOcarbamoyl carbamoyl phosphate Pi NH3+ + H3N-CH2CH2CH2CH-CO2- ornithine NH3+ O H2N-C-NH-CH2CH2CH2CH-CO2+ H+ citrulline This step also occurs in the mitochondrial matrix. 5 Combination of citrulline with aspartate to form argininosuccinate is driven by breakdown of ATP to AMP CO2O2C-CH2CH-NH3+ aspartate NH3+ O H2N-C-NH-CH2CH2CH2CH-CO2ATP citrulline AMP + PPi + H2O argininosuccinate NH2+ CO2NH3+ O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2This This reaction occurs only in the cytosol, so citrulline first must leave the mitochondria. A transporter exchanges ornithine for citrulline plus a proton across the mitochondrial inner membrane. 6 Argininosuccinate splits into arginine and fumarate NH3+ CO2- NH2+ O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2argininosuccinate O2C-CH=CH-CO2- - fumarate NH2+ NH3+ H2N-C-NH-CH2CH2CH2CH-CO2arginine This reaction occurs in the cytosol. This 7 Hydrolysis of arginine releases urea and regenerates ornithine NH3+ NH2+ H2N-C-NH-CH2CH2CH2CH-CO2arginine H2O O H2N-C-NH2 urea NH3+ H2N-CH2-CH2-CH2-CH-CO2H+ ornithine This This reaction occurs in the cytosol. To continue the cycle, ornithine must return to a mitochondrion. 8 HCO3- 2 ATP 2 ADP + Pi OO H2N-C-O-P-OO- NH4+ urea ATP 2 Pi arginine Pi citrulline NH3+ O H2N-C-NH-CH2CH2CH2CH-CO2- ornithine O H2N-C-NH2 carbamoyl phosphate PPi + AMP CO2O2C-CH2CH-NH3+ Asp H2O CO2- NH2+ NH3+ O2C-CH2CH-NH-C-NH-CH2CH2CH2CH-CO2argininosuccinate argininosuccinate Formation of urea consumes 4 phosphate anhydride bonds 9 The aspartate consumed in the urea cycle can be regenerated from the fumarate that is produced 2 ATP 2 ADP + Pi carbamoyl phosphate HCO3- + NH4+ α-keto acids amino acids Pi aspartateoxaloacetate aminotransferase ornithine citrulline Urea cycle urea ATP AMP + PPi arginine oxaloacetate aspartate argininosuccinate fumarate malate dehydrogenase NADH malate NAD+ H2O This process also uses both cytosolic and mitochondrial enzymes 10 Oxidation of malate in mitochondria generates ATP 2 ATP 2 e- to O2 via NADH dehydrogenase generates ~ 2.5 ATP 2 ADP + Pi carbamoyl phosphate HCO3+ NH4+ mitochondrion Pi ornithine citrulline ATP urea glutamate aspartate α-ketoglutarate citrulline ornithine oxaloacetate α-ketoglutarate NADH NAD malate glutamate aspartate AMP + PPi arginine cytosol cytosol argininosuccinate fumarate amino acids α -ketoacids malate H2O The mitochondrial inner membrane has transporters for malate, aspartate, glutamate and α-ketoglutarate, but not for NADH, NAD+ or oxaloacetate. 11 Transport systems in the mitochondrial inner membrane exchange aspartate for glutamate and α -ketoglutarate for malate mitochondrion aspartate- aspartate- glutamate- + H+ glutamate- + H+ α-ketoglutarate α-ketoglutarate malate malate cytosol The Asp/Glu transporter can be driven by an electrochemical potential gradient because it also moves a proton across the membrane. Mutations in this transporter have been linked to autism. 12 Autism is a neurodevelopmental genetic disorder Deficits in verbal & nonverbal communication and social interactions Repetitive or stereotyped behaviors Incidence ~1 per 1000 people (possibly 1 in 200) Strong evidence for heritability Polygenic - between 5 & 10 genes may be involved Mutations of neuronal cell-adhesion molecules (cadherins) implicated K. Wang et al., Nature 459: 528 (2009) Single-nucleotide polymorphisms (SNPs) in the gene for a mitochondrial, Ca2+-dependent Asp/Glu exchanger increase the risk by a factor of 3 to 4. This is the main form of the Asp/Glu exchanger that is expressed in the brain. Mutations in the gene impair the urea cycle, and could have other effects on mitochondrial metabolism. N. Ramoz et al., Am. J. Psychiatry 161: 662 (2004) L. Palmieri et al., EMBO J. 20: 5060 (2001) 13 The α-ketoglutarate/malate and aspartate/glutamate transporters also participate in oxidation of cytosolic NADH mitochondrion mitochondrion NAD+ NADH 2 e- to electrontransport chain oxaloacetate aspartate aspartate glutamate glutamate α-ketoglutarate α-ketoglutarate malate malate oxaloacetate cytosol cytosol NADH NAD+ glycolysis 14 The E. coli lactose permease provides a model for other small-molecule transporters TDG The entry to the cavity is open on this side cytosol membrane periplasm Lac Lac permease transports a proton along with a βgalactoside across the plasma membrane. TDG = 1-thio-β-D-galactopyranoside 12 trans-membrane α-helices surround a hydrophilic cavity closed on this side J. Abramson et al., Science 301: 610 (2003) 1pv7.pdb 15 The mitochondrial ATP/ADP exchanger (translocase) also has a central cavity that opens to the solution on one side of the membrane atractyloside (an inhibitor of ATP/ADP exchange) 6 trans-membrane α-helices surround a cavity view normal to the membrane surface H. Nury et al., FEBS Lett. 579: 6031 (2005) 2c3e.pdb The The ATP/ADP translocase is the most abundant protein in the mitochondrial inner membrane. It carries a proton into the matrix with each ATP-ADP exchange. 16 The urea cycle is regulated in two ways 1. Allosteric activation of carbamoylphosphate synthetase-I by N-acetylglutamate CO2+ H3N C H CH2 Glu arginine + acetyl-CoA CO2CH3CO-NH C H N-acetylglutamate CH2 CH2 - CoA-SH CH2 CO2 CO2- • Carbamoylphosphate synthetase-I is completely inactive in the absence of N-acetylGlu. • N-acetylGlu synthase is activated by arginine. • A genetic deficiency in N-acetylGlu synthase can cause a lethal defect in the urea cycle. • A specific hydrolase removes N-acetylGlu. NH4+ + HCO3- 2 ATP 2 ADP + Pi carbamoylphosphate OO H2N-C-O-P-OO- 2. A high-protein diet or starvation leads to increased synthesis of all five enzymes used in the urea cycle, including carbamoylphosphate synthetase-I. Expression of N-acetylglutamate synthase also increases. 17 Inherited disorders of the urea cycle can have severe effects Defective enzyme Effects Incidence* carbamoylphosphate synthetase-I lethargy, convulsions, early death < 0.5 argininosuccinate lyase argininosuccinic acidemia, vomiting, convulsions 1.5 arginase arginemia, intellectual disability < 0.5 *Approximate incidence per 100,000 births People with these conditions can’t be treated by simply removing proteins from their diet because humans are not able to synthesize all the necessary amino acids. 18 Genetic deficiencies in some of the urea-cycle enzymes can be treated pharmacologically benzoate CO2- CO2- ATP + CoA-SH AMP + PPi ATP + CoA-SH AMP + PPi O benzoyl-CoA hippurate (benzoylglycine) S-CoA S-CoA O glycine CoA-SH phenylacetate phenylacetyl-CoA glutamine CoA-SH O N H CO2- H N CO2- phenylacetylglutamine O O NH2 The amide products of these reactions (hippurate and phenylacetylglutamine) are excreted in the urine. Synthesizing the Gly or Gln removes ammonia. 19 Arginine also serves as a precursor of nitric oxide (NO) arginine NH3+ NH2+ H2N-C-NH-CH2CH2CH2CH-CO2NADPH, O2 Hydroxyarginine (bound to enzyme) NADP+, H2O NH3+ N-OH H2N-C-NH-CH2CH2CH2CH-CO2- The enzyme nitric oxide synthase, which catalyzes both steps, has four bound cofactors: FMN, FAD, heme and tetrahydrobiopterin 1/2 NADPH, O2 NO• citrulline 1/2 NADP+, H2O O H2N-C-NH-CH2CH2CH2CH-CO2- I won’t expect you to remember these details or the structure of hydroxyarginine. NO acts as a short-lived messenger in control of blood pressure, blood clotting, and NO neurotransmission. It binds to a guanylyl cyclase and activates production of c-GMP. 20 leucine lysine phenylalanine tryptophan tyrosine isoleucine leucine tryptophan pyruvate alanine cysteine glycine serine tryptophan The carbon chains of the common amino acids provide materials that feed into the citric acid cycle acetoacetyl-coA isocitrate acetyl-coA glutamate citrate arginine glutamine histidine proline α-ketoglutarate oxaloacetate succinyl-CoA isoleucine methionine threonine valine succinate asparagine aspartate malate fumarate phenylalanine tyrosine 21 leucine lysine phenylalanine tryptophan tyrosine isoleucine leucine threonine tryptophan pyruvate alanine cysteine glycine serine threonine tryptophan ketogenic Amino acids can be classified as glucogenic or ketogenic Some are in both groups acetoacetyl-coA isocitrate acetyl-coA glutamate citrate arginine glutamine histidine proline α-ketoglutarate oxaloacetate succinyl-CoA isoleucine methionine threonine valine succinate asparagine aspartate malate fumarate glucogenic phenylalanine tyrosine 22 Humans can synthesize 10 of the 20 common amino acids Amino acids that an organism cannot synthesize in adequate amounts under a given set of conditions are called “essential.” Those that can be synthesized in adequate amounts are “nonessential.” Most bacteria and plants can synthesize all 20 amino acids. Essential and Nonessential Amino Acids for Humans Essential Arginine* Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Nonessential Alanine Asparagine Aspartate Cysteine Glutamate Glutamine Glycine Proline Serine Tyrosine *Arg is essential in infants and growing *Arg children but not in adults. 23 Building blocks for synthesis of non-essential amino acids in humans come from glycolysis and the citric acid cycle glucose 3-phosphoglycerate glycine serine cysteine pyruvate alanine proline citrate oxaloacetate aspartate α -ketoglutarate glutamate glutamine arginine asparagine 24 Serine is formed via 3-phosphoglycerate 1 CO2H-C-OH CH2-O- P 3-phosphoglycerate CO2C=O CH2-O- P NAD+ NADH 3-phosphohydroxypyruvate Glu α -kG CO2+ H3N-C-H CO2+ H3N-C-H CH2-OH serine CH2-O- P Pi H2O 3-phosphoserine Expression of enzyme 1 (phosphoglycerate dehydrogenase) is elevated in some cancer cells and is a potential target for cancer therapy. These cells appear to require the serine pathway in order to generate high levels of α-ketoglutarate from glutamate and glutamine [R. Possemato et al. Nature 476: 346 (2011)]. 25 ...
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This note was uploaded on 02/17/2012 for the course CHEM 212 taught by Professor Staff during the Fall '10 term at Rutgers.

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