Amino Acid Catabolism - Protein Catabolism

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 Degradation (Catabolism) Exam-3 material Fat Catabolism Digestion diet storage Inside of cells Fatty acid Catabolism Protein turnover FOUR stages in the catabolism of lipids: Ubiquitin Mobilization from tissues (mostly adipose) hormone regulated specific lipases glycerol Activation of fatty acids Fatty-acyl CoA Synthetase Transport carnitine Oxidation Proteosome Amino-Acid Degradation Dealing with the nitrogen Ammonia free transamination Urea Cycle Rationale Saturated FA Dealing with the carbon b-oxidation 4 steps: dehydrogenation, hydration, oxidation, thiolase energetics Unsaturated FA energetics Odd-chain FA Ketone Bodies Other organelles Protein Catabolism Protein Turnover (within cell): THE 26S PROTEOSOME 19S = a6b3g10 Chymotrypsinlike 20S = a7b14a7 19S = a6b3g10 20S does the protease. The b subunits are isozymes; proteases with different specificities 19S does ATP hydrolysis (a), Ubx recognition (g), and unfolding (g). 1 Protein Catabolism Rpn-10 ubiquitin receptor Rpn-11 de-ubiquinating enzyme • Cryo-EM was used to attain these structures within the last 5 years. ATPase hexamer Protein Catabolism Fate of Amino Acids: Catabolism Anabolism Protein Biosynthesis Oxidation Once broken down to amino acid, all types of protein are treated the same way depending on the organism’s energy needs: 1. Recycled into new proteins 2. Oxidized for energy – There are two separate aspects: o removal of amino group (urea cycle) o entry into central metabolism (glycolysis, citric acid cycle) We’ll call this “Amino Acid Degradation or Catabolism” 2 Amino Acid Catabolism Nitrogen Catabolism Protein catabolism Glycogenolysis Amino-acid catabolism Fat Catabolism Fatty Acid Glycolysis Degradation Pyruvate Oxidation Krebs' Cycle Phosphorylation Oxidative 3 Amino Acid Catabolism Step 1: Removal of the Amino Group • Release of free ammonia, which is toxic. • Instead of Ammonia, nitrogen is captured by a series of trans-aminations (TA). • Transaminations allow transfer of an amine to a common metabolite (e.g., α-ketoglutarate) and generate a traffickable amino acid (e.g., glutamate). Cellular protein TA • Catalysis by aminotransferases (TA): • Uses the pyridoxal phosphate (PLP) cofactor • Typically, a-ketoglutarate accepts amino groups. – Transfer of one amine to a-ketoglutarate results in synthesis of glutamate (e.g., transamination). – Transfer of a second amine results in synthesis of glutamine (e.g., glutamine synthetase). • L-Glutamine acts as a temporary storage of nitrogen and a way of de-toxifying the ammonia • L-Glutamine can donate the amino group when needed for amino acid biosynthesis. TA Amino Acid Catabolism Catabolism Anabolism Nucleotides a- + NH4 2° products CO2 a-Keto acids [N,Q,H(2),S,T,G,M,W(I)] a-Ketoglutarate Aspartate Glutamate Sugars Oxaloacetate Fatty acids 4 Amino Acid Catabolism What is the fate of the free ammonia and Asp? a-Amino groups Ammonia UREA CYCLE Carbon dioxide UREA Amino Acid Catabolism: Transamination* • Catalyzed by aminotransferases* • Uses the pyridoxal phosphate cofactor • Typically, either oxaloacetate or aketoglutarate accept amino groups. – Transfer of the a-amino group to aketoglutarate results in synthesis of glutamate (e.g., transamination). Vitamin B6 Ala | PLP *same thing Pyr aKG (E·PMP) Ping-pong bi bi Glu | PLP L-Alanine Pyruvate 5 Amino Acid Catabolism: Transamination Vitamin B6 Structure of Pyridoxal Phosphate and Pyridoxamine Phosphate • Intermediate, enzyme-bound carrier of amino groups • The linkage is made via a nucleophilic attack of the amino group of an active-site lysine. • Making an internal aldimine • Aminated form can react reversibly with carbonyl groups to make a Schiff base. • Aldehyde form can react reversibly with amino groups to make a Schiff base…… which is how its attached to the enzyme as a prosthetic group. Amino Acid Catabolism: Transamination 6 Amino Acid Catabolism: Transamination See Sapling animated Figure(a) (protonation of aldimine-carbon) E-PMP Aromatic Quinoid http://media.saplinglearning.com /priv/he/lehninger/aem/1806a_pyridoxal_phosphate.html Amino Acid Catabolism: Urea Cycle Synthesis of Carbamoyl Phosphate ① ② ⑤ UREA CYCLE ③ ④ Carbon dioxide 7 Amino Acid Catabolism: Urea Cycle ① 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). Amino Acid Catabolism: Urea Cycle of Carbamoyl ① Synthesis Phosphate Ammonia b Ammonia travels Carbamate travels Carbamoyl a Phosphate Carbamoyl Phosphate Synthetase II (ab)3 N-acetylglutamate Carbamoyl Phosphate Synthetase I 8 Amino Acid Catabolism: Urea Cycle The Urea Cycle ① ② ⑤ UREA CYCLE ③ ④ 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 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 9 Amino Acid Catabolism: Urea Cycle 10