SCBC203 Amino Acid Metabolism

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1 Breakdown of proteins Route I: Dietary protein breakdown SCBC203 Amino Acid Metabolism Dr Sarawut Jitrapakdee Professor of Biochemistry Department of Biochemistry Faculty of Science Mahidol University 1 Mostly transported and stored in muscle 2 Breakdown of proteins Route 2: Muscle protein breakdown (prolonged starvation or excessive exercise) Catabolism of amino acids 3 Amino acids are not normally catabolized during feeding period Amino acids are used for protein synthesis during feeding period (anabolic) Amino acid catabolism will occur during starvation and excessive exercise Amino acids catabolism occurs mainly from muscle protein and small amounts from food. Catabolism of amino acids occurs in both liver and muscle 4

2 Concept of amino acid catabolism Amino acids from proteins are: - precursors of compounds - energy source (i.e., converted to acetyl-coa, etc.) Removal of amino group Trasamination: -NH2 is transferred to acceptor molecule Amino acids are obtained in diet and/or turnover of cellular proteins. Major problem in amino acid degradation is elimination of amino group (-NH 2 ) since NH 3 from NH 2 is very toxic Ammonia eliminations are: - Conversion to urea (mammals) - Conversion to uric acid (birds) Oxidative deamination: -NH 2 is oxidized Carbon skeleton of amino acid metabolism is:- NH 2 group is removed by transamination & oxidative deamination to urea. 5 6 Transamination Removal of amine from amino acids Keto acid is used as NH 2 acceptor and becomes second amino acid Once donated NH 2, amio acid 1 becomes keto acid Pyridoxal phosphate (B6) is a cofactor of reaction. 7 8

3 Urea cycle (overall) Reactions in Urea Cycle Liver ONLY mitochondria + cytoplasm NH 3 + HCO 3 - Produces urea and fumarate Fumarate enters TCA cycle forming Krebs bicycle Enzymes in urea cycle Urea (soluble waste) 9 10 Cooperation of Urea cycle and Citric Acid Cycle (Krebs bicycle) Regulation of urea cycle Protein breakdown KREBS BICYCLE Fumarate from urea cycle enters krebs cycle. Asparate is formed from -ketoglutarate in krebs cycle enters urea cycle. 11 Regulatory step: Carbamoyl phoshate synthetase (CPSI) CPSI is stimulated by NAG High NAG, high activity of CPS, high urea cycle activity 12

4 Muscle does not have urea cycle Route 2: Converts to alanine in glucose-alanine cycle How does it get rid off ammonia? Route 1: Converts to Glutamine Glutamate dehydrogenase Conversion of carbon skeleton of amino acids Conversion of carbon skeleton of amino acids 20 amino acids are converted to 7 common intermediates 1. Pyruvate Glucogenic amino acids * * * -ketoglutarate 3. Succinyl-CoA 4. Fumarate 5. Oxaloacetate Glucogenic amino acids * * * * 6. Acetyl-CoA Ketogenic amino acids 7. Acetoacetate Glucogenic amino acids -> Glucose (via gluconeogenesis) Ketogenic amino acids -> ketone body 15 * * * * * Both 16

5 Example of catabolism of carbon skeleton of amino acids Arg, Pro, His, Gln Catabolism of branched chain amino acids (BCAA) Val, Leu, Ile 17 Regulated by BCAT and BCKDH enzymes Produces acetyl-coa or succinyl-coa 18 Example of catabolism of carbon skeleton of amino acids Amino acid synthesis 19 Occurs in skeletal muscle and liver Synthesize from glycolytic and TCA cycle intermediates Occur during feeding period (glycolysis + high TCA cycle) 20

6 Amino acid synthesis Synthesis of Ala, Asp, Asn, Glu and Gln Amino acids are not only the components of proteins, but also precursors to various compounds i.e. neurotransmitters, hormone and porphyrins. Animal lacks pathways to synthesize essential amino acids. 21 Transamination to keto acid intermediates (pyruvate, oxalocaetate, -ketoglutarate) 22 Synthesis of Gly, Ser, Cys Synthesis of Arg and Pro Glycolytic intermediate Glutamate donates NH 2 Folate (remove Carbon) -SH in Cys is from SO Glutamate is a starter Requires NADPH+H Urea cycle 24

7 Heme biosynthesis Synthesis of hormone and neurotransmitters Succinyl-CoA Glycine donates amine group Synthesis of glutathione (anti-oxidant protein) SCBC203 Integrate Metabolism Dr Sarawut Jitrapakdee Professor of Biochemistry Department of Biochemistry Faculty of Science Mahidol University 27 28

8 Plasma glucose Plasma glucose is maintained via actions of glucagon and insulin Starvation glycerol FFA pancreas glucagon adipocyte myocyte RBC + + amino acid gluconeogenesis glycogenolysis + glucagon vs insulin action lactate Brain Feeding + De novo fat synthesis glycolysis glycogenesis + De novo fat synthesis adipocyte (TG) pancreas insulin + glycogenesis myocyte - RBC Brain Metabolic adaptation during starvation Glycogen breakdown: to release glucose Muscle protein breakdown and convert to glucose (glucose alanine cycle gluconeogenesis) Triglyceride mobilization from adipose tissue to liver and skeletal muscle for ( oxidation) Glycerol from triglyceride is used for gluconeogenesis Fatty acid oxidation Ketone synthesis (excrete to brain) Gluconeogenesis to supply glucose for muscle, RBC and brain glucose glucose 31 32

9 Glycogen breakdown during starvation Glucose-alanine cycle G6Pase Glucose from glycogen cannot release outside muscle Because muscle does not have glucose 6 phosphatase (G6Pase) 33 Glucose alanine cycle: Starvation (no glucose) Protein is converted to amino acid (alanine) Alanine is converted to glucose (gluconeogenesis) in liver Liver releases glucose back to muscle 34 Glucose produced by gluconeogenesis also use as energy for brain and red blood cells During starvation Fat is transported from adipose tissue to muscle and liver for beta oxidation (reduce the use of glucose as fuel) Starvation/exercise Glucagon/catecholamine Substrates for gluconeogenesis 1. Lactate from RBC, muscle 2. Alanine from muscle protein 3. Glycerol from adipose tissue 35 36

10 Excessive oxidation produces ketone body in liver Brain, heart, Muscle Can oxidize ketone during starvation Muscle uses different nutrients during different light and heavy exercise Lactate produced during heavy exercise is transported to liver and converted to glucose by gluconeogenesis 39 Recycle of lactate to glucose by gluconeogenesis and returning of glucose from liver to muscle forms Cori cycle 40

11 Metabolic adaptation during feeding (plenty of food) Glycogen synthesis in muscle and liver De novo fatty acid synthesis (liver &adipose tissue) Muscle protein synthesis (Muscle) Glycogenolysis Gluconeogenesis - Glycolysis Glycogenesis Lipogenesis LIVER + METABOLIC EFFECTS OF INSULIN Glucose - + insulin Glycogenolysis Protein breakdown GLUT4 Glycogenesis Protein synthesis MUSCLE GLUT4 Lipolysis Lipogenesis LPL ADIPOCYTES 42 Glycogen metabolism (liver & muscle) Insulin stimulates glycolysis and de novo fatty acid enzymes De novo fatty acid synthesis in liver Insulin inactivates glycogen synthase kinase 3 (GSK3) Glycogen synthase can function (normally suppressed by GSK3) Excess glucose is diverted to de novo fatty acid synthesis

12 SCBC203 Nucleotide Metabolism Insulin stimulates ribosome biogenesis and elongation Factors, eef4e, eef2 activities 45 Dr Sarawut Jitrapakdee Professor of Biochemistry Department of Biochemistry Faculty of Science Mahidol University 46 Nucleotide Metabolism Oxidation (Catabolism) Purine catabolism Pyrimidine catabolism Nucleotide structure Synthesis (anabolism) Salvage pathway De novo Pathway 47 Nucleotide = Nitrogenous base ribose sugar phosphate Nucleotides found in dietary meat product 48

13 Facts about nucleotide catabolism Catabolism of purine (A,G) Occur in liver to produce base, ribose and phosphate Bases are catabolized in liver to produce uric acid Excess bases and ribose are recycled for synthesis of nucleotides in muscle, brain or dividing cells (need to synthesize nucleic acids) [Salvage pathway, see later] Nucleotide catabolism occurs in mitochondria Do not produce ATP Gout is caused by consuming too much meat product 49 Order of reaction Phosphate is removed first (nucleotidase) Ribose is removed later (purine nucleotide phosphorylase, PNP) Xanthine oxidase is the final regulatory enzyme Uric acid is the final product of purine catabolism Catabolism of Pyrimidine (C,T,U) cytosine 50 Deamination Urea cycle Urine uric acid crystals Overload of nucleotide in purine catabolic pathway Excessive conversion of purine to uric acid Uric acid precipitated in joint and causes joint pain Allopurinol inhibits xanthine oxidase and reduces production of uric acid 51 Difference from Purine Catabolism Requires NADPH+H Unlike purine catabolism, pyrimidine ring is cleaved and yields CO 2 and NH 3 (complete oxidation) 52 NH 3 enters urea cycle

14 De novo vs Salvage pathways Nucleotide biosynthesis 53 De novo = synthesis from sugar and amino acids Salvage = Synthesis from Degradation of nucleic acid (N base + ribose + phosphate) 54 De novo pathway 1. Synthesis of ribose sugar: pentose phosphate pathway 55 56

15 De novo pathway 2. Formation of phosphoribosyl pyrophosphate (PRPP) 3. Purine synthesis (A, G) De novo pathway Catalyzed by enzyme Phosphoribosyl pyrophosphate synthetase Two phosphate are attached at the C1 of pentose forming two pyrophosphate PRPP is an activated ribose sugar 57 Common intermediate is inosine monophosphate (IMP) IMP is further converted to AMP and GMP 58 Things to remember NDP is further converted to dndp by Ribonucleotide Reductase (RR) Nitrogen comes from Asp, Gly,Gln 2C is form tetrahydrofolate 1C is from CO 2 59 OH at C 2 is converted to H 60

16 De novo pathway 3. Pyrimidine synthesis (C, T) CDP is further converted to dndp by Ribonucleotide Reductase (RR) Nitrogen comes from Gln, Asp 3C is from Asp 1C is from CO 2 Orotic acid is the common product Conversion of dump to dtmp requires folate as a co-factor 5-fluocaril and Methotrexate block dihydrofolatetetrahydrofolate regeneration (block dtmp synthesis) C is transferred to U NADP + NADPH + H C is removed from CH 2 Serine Three enzymes works together to Regenerate tetrahydrofolate into three forms (di-, tri-, tetra-) Thymidylate synthase (TS) Dihyrofolate reductoase (DHFR) Serine hyroxymethyltransferase (SHMT) 63 Methotrexate (kills cancer, T-cell (rheumatoid), anti-malaria) 5-fluorouracil 64

17 Regulation of nucleotide synthesis Purine Salvage pathway DNA/RNA degradation Regulatory steps (Product inhibition-high levels of IMP, AMP, GMP) Phosphoribosylamine synthesis Conversion of IMP to AMP and GMP HPRT reaction 65 Recycle N-base (A,G) from DNA/RNA degradation and couple with phosphoribosyl pyrophosphate (PRPP) to form AMP or GMP 66 Catalyzed by enzyme Hypoxanthine-Guanine phosphoribosyl transferase (HPRT) Defect of HPRT causes genetic disease Lesch-Nyhan Syndrome X 67 Accumulation of hypoxanthine+guanine Hypoxanthine and Guanine diverged 68 toward uric acid production (similar to Gout)

18 Pyrimidine Salvage pathway De novo synthesis enzyme UPRT PRPP + Uracil UMP UDP dudp dtmp CDP dcdp CTP Uracil phosphoribosyl transferase (UPRT) 69