BI/CH 422/622 OUTLINE: OUTLINE: Protein Degradation (Catabolism) Digestion Amino-Acid Degradation Inside of cells Urea Cycle – dealing with the nitrogen Protein turnover Ubiquitin Feeding the Urea Cycle Activation-E1 Glucose-Alanine Cycle Conjugation-E2 Free Ammonia Ligation-E3 Proteosome Glutamine Amino-Acid Degradation Glutamate dehydrogenase Ammonia Overall energetics free Dealing with the carbon transamination-mechanism to know Seven Families Urea Cycle – dealing with the nitrogen 1. ADENQ 5 Steps 2. RPH Carbamoyl-phosphate synthetase oxidase Ornithine transcarbamylase one-carbon metabolism Arginino-succinate synthetase THF Arginino-succinase SAM Arginase 3. GSC Energetics PLP uses Urea Bi-cycle 4. MT – one carbon metabolism 5. FY – oxidases 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 a-amino acid Aspartate transaminase transaminase a-keto acid Glutamate 1 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 acetyl- CoA), 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 Amino Acid Catabolism: Urea Cycle Excess glutamine is processed in the intestines, kidneys, and liver. (deaminating) (N,Q,H,S,T,G,M,W) OAA à Asp Glutamine Synthetase This costs another ATP, bringing it closer to 5 (N,Q,H,S,T,G,M,W) 29 N 2 Amino Acid Catabolism: Urea Cycle Excess glutamine is processed in the intestines, kidneys, and liver. (deaminating) (N,Q,H,S,T,G,M,W) OAA à Asp Glutamine Synthetase This costs another ATP, bringing it closer to 5 (N,Q,H,S,T,G,M,W) Amino Acid Catabolism: Urea Cycle Regulation of the Glutamate Urea Cycle Urea Cycle Dehydrogenase + NADP+ H NADPH 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 Schiff base between catabolic and anabolic pathways; formation when biosynthesis is needed, its off and Glu provides for amino-acid biosynthesis. When energy is needed, the ammonia is • GluDH has a high K value (>1 mM) for ammonia, so pulled off to provide for the urea cycle and m it mostly functions to provide the initial material for the oxidation of carbon skeletons. urea cycle; ammonia, using NAD+, which is high when • Can use either NAD+ in the catabolic energy charge is low. direction (oxidative deamination), or • Ammonia is processed into urea for excretion. Carbon NADPH in the biosynthetic direction. skeletons are oxidized for ATP. 3 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 N-acetyl- concentrations are high glutamate –Km values are 1 and 0.7 mM, respectively –activated by arginine (Ki value of 10 µM!) •Expression of urea cycle enzymes increases when needed. –high-protein diet –starvation, when protein is being broken down for energy 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. • Humans and great apes excrete both Ure-otelic urea (from amino acids) and uric acid (from purines). Uric-otelic 4 Amino Acid Catabolism: Urea Cycle Energetics of Urea Cycle & Amino acid degradation Example: 2 Alanines GluDH NADP+ à NADPH 2.5 MDHcyto Pyr NAD+ à NADH 2.5 Ala 5 Per Pyruvate in Krebs Cycle: 4 NAD+ à 4 NADH 10 FAD à FADH2 1.5 GTP 1 aKG 12.5 x 2 = 25 Ala 25 + 5 = 30 – 4 to 5 (Urea Cycle) Pyr Glu ~13 ATP per Ala Amino Acid Degradation: the carbon “skeletons” A. Concepts 1. Convergent 2. ketogenic/glucogenic The SEVEN (7) Families 3. Reactions seen before B. Transaminase (A,D,E) / Deaminase (N,Q) Family C. Related to biosynthesis (R,P,H; G,S,C; T,M) 1.Glu Family a. Introduce oxidases/oxygenases b. Introduce one-carbon metabolism (1C) 2.Pyruvate Family a. PLP reactions 3. a-ketobutyric Family a. 1-C metabolism D. Dedicated (F,Y; K,W; V,I,L) 1. Aromatic Family a. oxidases/oxygenases 2. a-ketoadipic Family 3. Branched-chain Family 29 N 5 Amino Acid Degradation • Intermediates of the central metabolic pathway • Some amino acids result in more than one intermediate. • Ketogenic amino acids can be converted to ketone bodies. Seven to Acetyl-CoA Leu, Ile, Thr, Lys, Phe, Tyr, Trp • Glucogenic amino acids can be converted to glucose. Six to pyruvate1 Ala, Cys, Gly, Ser, Thr, Trp Five to a-ketoglutarate2 Arg, Glu, Gln, His, Pro Four to succinyl-CoA Ile, Met, Thr, Val Two to fumarate3 Phe, Tyr Two to oxaloacetate4 Asp, Asn 1Pyruvate family 2Glu family 3Aromatic family 4Trans-/de-aminase family Amino Acid Degradation A,D,E Simple Transamination PLP PLP + + PLP 6 Amino Acid Degradation N,Q Simple Deamination PLP + + PLP Amino Acid Degradation A,D,E,N,Q • Intermediates of the central metabolic pathway • Some amino acids result in more than one intermediate. • Ketogenic amino acids can be converted to ketone bodies. Seven to Acetyl-CoA Leu, Ile, Thr, Lys, Phe, Tyr, Trp • Glucogenic amino acids can be converted to glucose. Six to pyruvate1 Ala, Cys, Gly, Ser, Thr, Trp Five to a-ketoglutarate2 Arg, Glu, Gln, His, Pro Four to succinyl-CoA Ile, Met, Thr, Val Two to fumarate3 Phe, Tyr Two to oxaloacetate4 Asp, Asn 1Pyruvate family 2Glu family 3Aromatic family 4Trans-/de-aminase family 7 Amino Acid Degradation R,P,H Glu Family Arginine degradation is part of the urea cycle. PLP Proline, arginine, and histidine are all converted to glutamate. Histidine-ammonia lyase uricanase Imidazolone- propionase Glu-formiminotransferase Histidine Urocanate Imidazolone- Formimino propionate glutamate Two new metabolic concepts used: 1) Oxidase 2) One-carbon metabolism N 5N 10-Methenyl N 5-Formimino THF THF Amino Acid Degradation R,P,H Glu Family Arginine degradation is part of the urea cycle. PLP Proline, arginine, and histidine are all converted to glutamate. Histidine-ammonia lyase uricanase Imidazolone- propionase Glu-formiminotransferase Histidine Urocanate Imidazolone- Formimino propionate glutamate Two new metabolic concepts used: 1) Oxidase 2) One-carbon metabolism N 5N 10-Methenyl N 5-Formimino THF THF 8 Amino Acid Degradation: One Carbon H H H • Tetrahydrofolate is formed H from folate. dihydro – an essential vitamin – Folate is reduced to dihydrofolate, then to tetrahydrofolate (THF) • It is used in a wide variety of metabolic reactions. • THF can transfer 1-carbon in different oxidation states. – CH3, CH2OH, and CHO • Carbon generally comes from serine. • Forms are interconverted on THF before use. 9.