Amino Acid Catabolism - Urea Cycle

OUTLINE: Introduction and review Transport Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic Exam-1 material Fermentations Pyruvate Krebs’ Cycle Oxidative Phosphorylation Exam-2 material Electron transport Chemiosmotic theory: Phosphorylation Protein Fat Catabolism Exam-3 material diet storage Fatty acid Catabolism FOUR stages in the catabolism of lipids: Degradation (Catabolism) Digestion Inside of cells Protein turnover Ubiquitin Mobilization from tissues (mostly adipose) Proteosome hormone regulated specific lipases Amino-Acid Degradation glycerol Dealing with the nitrogen Activation of fatty acids Fatty-acyl CoA Synthetase Ammonia Transport free carnitine transamination ß know mechanism Oxidation Carbamoyl-phosphate synthetase Rationale Saturated FA Urea Cycle b-oxidation 5 Steps 4 steps: dehydrogenation hydration oxidation thiolase Ornithine transcarbamylase Arginino-succinate synthetase Arginino-succinase Arginase energetics Unsaturated FA energetics Odd-chain FA Ketone Bodies Other organelles Energetics Urea Bi-cycle Dealing with the carbon Amino Acid Catabolism: Urea Cycle The Urea Cycle ① ② ⑤ UREA CYCLE ③ ④ 1 Amino Acid Catabolism: Urea Cycle The Urea Cycle: Evidence for a cycle - Already known that Arg gives rise to Urea and Orn ⑤ - How do CO2 and NH3 get into Arg? - Enter H. Krebs, who studies metabolism using the Warburg apparatus - Urease (recently purified by Sumner in 1925) can hydrolyzed urea to CO2 + 2 NH3; Could measure CO2 from urea by Warburg apparatus - Using liver slices, which amino acids gave rise to urea? - ….. Most gave some - Then as a control, he tried Orn - Added Orn to prep: extraordinary occurrence of “catalytic” amounts of urea!! Orn, which is the product, will give rise to more urea than the Orn added, and at a 7-30x increased rate: “catalytic” - In the library, Krebs looked for intermediates that might have CO2 & NH3 stuck to Orn: citrulline - Dr. Wada in Japan had just published the purification of citrulline from watermelon. -Krebs attained 10 mg citrulline and added it…...same catalytic phenomenon!! First cyclic process! …...lead to lots of notariety Dr. Kornberg: Lecture Amino Acid Catabolism: Urea Cycle The Urea Cycle ① ② ⑤ UREA CYCLE ③ ④ 2 Amino Acid Catabolism: Urea Cycle ① Recall: Synthesis of Carbamoyl Phosphate • The first nitrogen-acquiring reaction of the urea cycle Carbamoyl Phosphate Synthetase I • Excess CO2, ATP, and ammonia is present in liver mitochondria. This is where the activation of both waste products occur (the majority of the other urea-cycle reactions occur within the cytosol). • For step #2, in order to move to the cytosol, carbamoyl phosphate must condense with ornithine to create citrullene. This reaction releases the phosphate of carbamoyl phosphate into the mitochondrial matrix (so it does not deplete the proton motive force). Citrullene can then be transported to the cytosol. Amino Acid Catabolism: Urea Cycle ② Ornithine Transcarbamylase (OTCase) (in mitochondrial matrix) Recall: Aspartate Transcarbamoylase (ATCase) (a3)2(b2)3 3 Amino Acid Catabolism: Urea Cycle ③ Argino-succinate Synthetase • This is the second nitrogenacquiring reaction. • The synthetase is in the cytosol • citrulline reacts with ATP to produce citrullyl-AMP. • AMP acts as a good leaving group, as aspartate attacks the imide carbon to produce argininosuccinate. • PPi product helps drive reaction. Where have we seen this strategy before? What drives this reaction? http://media.saplinglearning.com/priv/he/lehninger/aem/1811b_argininosuccinate.html Amino Acid Catabolism: Urea Cycle ④ Argininosuccinase • Argininosuccinase cleaves fumarate from argininosuccinate, resulting in arginine. 4 Amino Acid Catabolism: Urea Cycle ④ Argininosuccinase ⑤ ② ① ③ ④ = make bond = break bond • Argininosuccinate is a good molecule to rationalize the cycle showing were and in what order bonds are made or broken. Amino Acid Catabolism: Urea Cycle ⑤ Arginase • Excess Arginine can also enter the urea cycle at this point. • Arginase cleaves both nitrogen atoms added in the urea cycle from ammonia and Asp, resulting in free urea. • Ornithine is able to serve as a substrate for the next round of the cycle. 5 Amino Acid Catabolism: Urea Cycle Enzymes of the Urea Cycle ① ② ③ ④ ⑤ Amino Acid Catabolism: Urea Cycle Energetics of Urea Cycle Per 2 nitrogen atoms to make one urea molecule: “energy of 3 ATP” à 2 Pi 3 To make CP (2) and arginosuccinate (1) 1 ATP 2ATP 2ADP, H+ , Pi Pi ATP Net is 4 ATP equivalents H2 O AMP, PPi [Its not actual ATPs used, its how many it takes to recycle the products (2 ADP + 1 AMP)] Ø But what about this fumarate? Ø What is its fate? Ø How do you regenerate Asp to keep the cycle going? 6 Amino Acid Catabolism: Urea Cycle Two issues: 1) What to do with the fumarate? 2) What are the sources of the free ammonia? Amino Acid Catabolism: Urea Cycle The Urea Bi-Cycle a-ketoglutarate Aspartate a-amino acid transaminase transaminase Glutamate a-keto acid 7 Amino Acid Catabolism: Urea Cycle The Urea Bi-Cycle Two issues: 1) What to do with the fumarate? 2) What are the sources of the free ammonia? a-ketoglutarate Aspartate a-amino acid transaminase transaminase Glutamate a-keto acid Amino Acid Catabolism: Urea Cycle The Glucose-Alanine Cycle • Vigorously working muscles operate nearly anaerobically and rely on glycolysis for energy. a -Keto acids • Glycolysis yields pyruvate. – If not eliminated (converted to acetylCoA), lactic acid will build up. • If amino acids have become a fuel source, this lactate is converted back to pyruvate, then converted to alanine for transport into the liver. Excess Glutamate is Metabolized in the Mitochondria of Hepatocytes (Asp) 8 Amino Acid Catabolism: Urea Cycle Excess glutamine is processed in the intestines, kidneys, and liver. (deaminating) (N,Q,H,S,T,G,M,W) O AA à Asp Glutamine Synthetase This costs another ATP, bringing it closer to 5/urea (N,Q,H,S,T,G,M,W) 29 N Amino Acid Catabolism: Urea Cycle Excess glutamine is processed in the intestines, kidneys, and liver. (deaminating) (N,Q,H,S,T,G,M,W) O AA à Asp Glutamine Synthetase This costs another ATP, bringing it closer to 5/urea (N,Q,H,S,T,G,M,W) 9 Amino Acid Catabolism: Urea Cycle Regulation of the Glutamate Urea Cycle Dehydrogenase Urea Cycle Important Example of oxidative deamination + N ADP + H N ADPH Allosteric inhibitors: GTP, ATP, Palmitoyl-CoA • The pool of Glu is a critical junction between biosynthesis and catabolism Allosteric Activators: ADP, Leucine, Isoleucine, Valine • The enzyme represents a key link between Schiff base catabolic and anabolic pathways; when form ation biosynthesis is needed, its off and Glu provides for amino-acid biosynthesis. When energy is needed, its on; the • GluDH has a high Km value (>1 mM) for ammonia, so ammonia is pulled off to provide for the it mostly functions to provide the initial material for the urea cycle and oxidation of carbon urea cycle; ammonia, using NAD+, which is high when skeletons. energy charge is low. + • Can use either NAD in the catabolic direction (oxidative deamination), or NADPH in the biosynthetic direction. • Ammonia is processed into urea for excretion. Carbon skeletons are oxidized for ATP. Amino Acid Catabolism: Urea Cycle Regulation of the Urea Cycle • Urea production is controlled by carbamoyl phosphate synthetase. • N-acetylglutamate is an allosteric activator. • Carbamoyl phosphate synthetase I is activated by N-acetylglutamate. • Formed by N-acetylglutamate synthase –when glutamate and acetyl-CoA concentrations are high –Km values are 1 and 0.7 mM, respectively –activated by arginine (Ka value of 10 µM!) N-acetylglutamate • Expression of urea cycle enzymes increases when needed. –high-protein diet –starvation, when protein is being broken down for energy 10 Amino Acid Catabolism: Urea Cycle Excretory Forms of Nitrogen: Fates of Nitrogen in Organisms • Plants conserve almost all the nitrogen. • Many aquatic vertebrates release ammonia to their environment. –passive diffusion from epithelial cells –active transport via gills • Many terrestrial vertebrates and sharks excrete nitrogen in the form of urea. –Urea is far less toxic that ammonia. –Urea has very high solubility. –Requires lots of water • Some animals such as birds and reptiles excrete nitrogen as uric acid. –Uric acid is rather insoluble. –Excretion as paste allows the animals to conserve water. Ure-otelic (urea) Uric-otelic (uric acid) • Humans and great apes excrete both urea (from amino acids) and uric acid (from purines). Amino Acid Catabolism: Urea Cycle Energy from Amino-Acid Degradation & Urea Cycle Example: 2 Alanines In liver GluDH NAD+ à NADH Pyr Ala 2.5 MDHcyto NAD+ à NADH 2.5 5 Per Pyruvate in Krebs Cycle: 4 NAD+ à 4 NADH 10 FAD à FADH2 1.5 GTP 1 12.5 x 2 = 25 aKG Ala Glu 25 + 5 = 30 – 4 (Urea Cycle) Pyr =26/2 = 13 ATP per Ala 11 Amino Acid Degradation: the carbon “skeletons” A. Concepts 1. Convergent 2. ketogenic/glucogenic The SEVEN 3. Reactions seen before B. Transaminase (A,D,E) / Deaminase (N,Q) Family (5) C. Related to biosynthesis (8) 1. Glu Family (R,P,H) a. b. (7) Families Introduce oxidases/oxygenases Introduce one-carbon metabolism (1C) 2. Pyruvate Family (G,S,C) a. PLP reactions a. 1-C metabolism In total there are 29 Nitrogen atoms in the 20 amino acids: 9 are given off as ammonia 18 are taken off in transaminase reactions 2 leave as urea There are ~75 reactions in total 3. a-Ketobutyric Family (M,T) D. Dedicated (7) 1. Aromatic Family (F,Y) a. oxidases/oxygenases 2. a-Ketoadipic Family (K,W) 3. Branched-chain Family (V,I,L) E. Convergence with Fatty Acids: propionyl-CoA 12