CHEM206 Week 7 - Protein and Amino Acid Metabolism PDF

CHEM206 Amino Acid Metabolism 1 | CHEM206 @ ACU 1 Amino Acid Metabolism Learning Objectives By the end of this topic you will understand : List the sources of amino aci...

CHEM206 Amino Acid Metabolism 1 | CHEM206 @ ACU 1 Amino Acid Metabolism Learning Objectives By the end of this topic you will understand : List the sources of amino acids for building proteins. Discuss nitrogen fixation The nitrogenase reaction Families of amino acids based on their biosynthetic pathways and the relationship between amino acids and the citric acid cycle Amino acid biosynthesis Describe what is meant by the term essential amino acids. Describe the breakdown of dietary proteins Discuss the process of protein turnover Explain the process whereby an amino group is removed form an amino acid. Discuss the degradation of AA in muscle during prolonged exercise and fasting. Describe how amino acid degradation is linked to the TCA cycle. Discuss the Urea cycle e.g. ATP used, where performed, where the two N in urea are from. Explain what is meant by the terms keto- and glucogenic amino acids and provide examples. Give examples of conditions where there are errors in the degradation of amino acids.. 2 | CHEM206 @ ACU 2 Nutrtion @ ACU 1  A question you have probably never asked: How do amino acids get their amino? 3 | CHEM206 @ ACU 3 Nitrogen Metabolism Consists of biosynthesis and breakdown of amino acids, purines, and pyrimidines Metabolism of porphyrins is related to that of amino acids Nitrification: Conversion of ammonia to nitrates Denitrification: Process by which nitrates and nitrites are broken down to molecular nitrogen 4 | CHEM206 @ ACU 4 Nutrtion @ ACU 2  Nitrogen Fixation Flow of Nitrogen in the Biosphere Nitrogen is the most common gas in the atmosphere - 78.09% (dry air) Issue: N N bond is very stable: bond energy = 225 kcal/mol Industrial: Haber-Bosch Process (CHEM105!) N2 + 3 H2 2 NH3 at 500°C, 300 ATM, Fe catalyst 5 | CHEM206 @ ACU 5 Nitrogen Fixation Flow of Nitrogen in the Biosphere Nitrogen is the most common gas in the atmosphere - 78.09% (dry air) Issue: N N bond is very stable: bond energy = 225 kcal/mol Biological: free-living soil bacteria - symbiotic bacteria in root nodules of legumes - cyanobacteria (i.e. blue-green algae) N2 + 8 e- + 8 H+ + 16 ATP 2 NH3 + 16 ADP + 16 Pi + H2 at biological temps, 0.8 ATM Redox Reaction 6 | CHEM206 @ ACU 6 Nutrtion @ ACU 3  Nitrogen Fixation Symbiotic bacteria inside root nodule cell Nitrogen Fixation Root nodules Catalyzed by the nitrogenase enzyme complex = 2 metalloproteins, “reductase” and “nitrogenase”: N2 + 8 e- + 8 H+ + 16 ATP 2 NH3 + 16 ADP + 16 Pi + H2 7 | CHEM206 @ ACU 7 Nitrogen Fixation Summary of the Nitrogenase Reaction N2 + 8 e- + 8 H+ + 16 ATP 2 NH3 + 16 ADP + 16 Pi + H2 8 | CHEM206 @ ACU 8 Nutrtion @ ACU 4  Nitrogen Fixation Summary of the Nitrogenase Reaction 2 Dinitrogenase Reductase: - contains Fe-S redox center, 3 6 oxidized/reduced by 1 e- transfers 1 4 - 1. receives e from Ferredoxin and transfers the e- to nitrogenase 2. 2 ATP hydrolyzed/e- transferred 5 3. ATP-binding enhances reducing power of the reductase (E’° -0.25 V  -0.4 V), which is required for e- transfer 4. ATP-binding causes conformational change that promotes binding of reductase to nitrogenase 5. ATP hydrolysis triggers e- transfer to nitrogenase and promotes dissociation 9 | CHEM206 @ ACU 9 6. Nitrogenase MoFe cofactor = site of N2 reduction Nitrogenase has an unusual redox center, a “cofactor” containing 1 Mo, 7 Fe, 9 S N N 2H+ + 2e- 6 HN NH + - 2H + 2e 5 2HN NH2 + - 2H + 2e 2 NH3 N2 believed to bind to Fe’s in central cavity of MoFe cofactor. Formation of multiple Fe-N interactions weakens the N N bond and lowers activation N2 reduction on the MoFe cofactor barrier for reduction occurs in 3 steps, & requires 6 e- The remaining 2 e- are used to reduce 2 H+ to H2 10 | CHEM206 @ ACU 10 Nutrtion @ ACU 5  NH4+ assimilation: glutamine & glutamate = entry points into metabolism Glutamate is formed by reductive amination of -ketoglutarate and NH4+ Amidation of glutamate gives glutamine Glutamate can also be formed by reversal of the glutamate dehydrogenase reaction (minor pathway) Involves transamination reactions and one-carbon transfers Transamination: Transfer of amino groups from one molecule to another 11 | CHEM206 @ ACU 11 Glutamate & Glutamine as N-donors Glutamate α-amino group is the source of the α-amino group in most other amino acids reactions catalyzed by aminotransferases (pyridoxal phosphate = cofactor) NOTE: this reaction is REVERSIBLE! Will come back for AA catabolism 12 | CHEM206 @ ACU 12 Nutrtion @ ACU 6  Amino Acid Biosynthesis Transamination Reactions Role of Pyridoxal Phosphate Biologically active form of vitamin B6 is pyridoxal phosphate (PyrP) PyrP - Participates in the catalysis of a wide variety of reactions of amino acids, including transaminations and decarboxylations Forms an imine (a Schiff base) with the - amino group of an amino acid Rearrangement gives an isomeric imine Hydrolysis of the isomeric imine gives an -ketoacid and pyridoxamine All reactions are reversible 13 | CHEM206 @ ACU 13 Glutamate & Glutamine as N-donors Glutamine donates its side-chain N (=amide N) in the biosynthesis of a wide range of compounds - reactions catalyzed by glutamine amidotransferases Two domains: 1. Hydrolysis of Glutamine NH3 passes through channel so it won’t get “lost” 2. NH3 reacts with acceptor substrate 14 | CHEM206 @ ACU 14 Nutrtion @ ACU 7  Classes of reactions involved in  synthesis 1. Transamination reactions (w. glutamate) 2. Transfer of amino groups derived from amide N of glutamine N5, N10 methylene THF 3. Transfer of 1 carbon groups using tetrahydrofolate as cofactor N N N 5 N CH2 N CH2 N CH2 H l 10 l l l l N- H3C N- H2C N- H From AA losing a Carbon group 15 | CHEM206 @ ACU 15 Allosteric Regulation of Glutamine Synthetase Activity by Feedback Inhibition Clearly, Glutamine and Glutamate are important for N-donation They need to be well controlled! Negative feedback or feedback inhibition 9 (!) inhibitors allosterically control this enzyme: Glycine, alanine, serine, histidine, tryptophan, CTP, AMP, carbamoyl phosphate and glucosamine-6- phosphate 16 | CHEM206 @ ACU 16 Nutrtion @ ACU 8  How are the 20 common amino acids synthesized? NH3：primarily donated by glutamate Building blocks Carbon Skeletons：α-keto acid 17 | CHEM206 @ ACU 17 Families of Amino Acids Based on Pathways Carbon skeletons come from intermediates in Glycolysis: PEP, pyruvate, 3-phosphoglycerate Pentose-P pathway: ribose-5-P, erythrose-4-P Citric acid cycle: oxaloacetate, -ketoglutarate 18 | CHEM206 @ ACU 18 Nutrtion @ ACU 9  Amino Acid Biosynthesis Relationship between Amino Acid Metabolism and the Citric Acid Cycle The CAC is amphibolic: Anabolic and catabolic 19 | CHEM206 @ ACU 19 Families of Amino Acids Based on Pathways Carbon skeletons come from intermediates in Glycolysis: PEP, pyruvate, 3-phosphoglycerate Pentose-P pathway: ribose-5-P, erythrose-4-P Citric acid cycle: oxaloacetate, -ketoglutarate 20 | CHEM206 @ ACU 20 Nutrtion @ ACU 10  Amino Acid Biosynthesis Biosynthesis of Serine (and of Glycine from Serine in all organisms) Transamination reaction in which glutamate is the nitrogen donor produces 3-phosphoserine and a -ketoglutarate Hydrolysis of phosphate group gives rise to serine Glycine is produced from serine by the transfer of a one-carbon unit to an acceptor 21 | CHEM206 @ ACU 21 Amino Acid Biosynthesis Biosynthesis of Serine (and of Glycine from Serine in all organisms) Glycine is produced from serine by the transfer of a one-carbon unit to an carbon acceptor - tetrahydrofolate 22 | CHEM206 @ ACU 22 Nutrtion @ ACU 11  Amino Acid Biosynthesis Conversion of Serine to Cysteine Source of sulfur in plants and bacteria differ from that in animals In plants and bacteria, serine is acetylated to form O-acetylserine Reaction is catalyzed by serine acyltransferase, with acetyl-CoA as the acyl donor Sulfur donor is 3′-phospho-5′-adenylyl sulfate 23 | CHEM206 @ ACU 23 Amino Acid Biosynthesis Methionine Cannot be produced in animals and must be obtained from dietary sources Essential amino acid as it cannot be synthesized by the body Reacts with ATP to form S-adenosylmethionine (SAM) when ingested SAM has a highly reactive methyl group This makes three single-carbon carriers we have now met. Can you name them? 24 | CHEM206 @ ACU 24 Nutrtion @ ACU 12  Amino Acid Biosynthesis Biosynthesis of Cysteine in Animals SAM is a methyl group carrier, and the methyl group can be transferred to a number of acceptors, producing S-adenosylhomocysteine Hydrolysis of S-adenosylhomocysteine produces homocysteine Cysteine can be synthesized from serine and homocysteine, and this pathway for cysteine biosynthesis is the only one available to animals 25 | CHEM206 @ ACU 25 Essential Amino Acids Essential Amino Acids     26 | CHEM206 @ ACU 26 Nutrtion @ ACU 13  Amino Acid Metabolism Learning Objectives By the end of this topic you will understand :  List the sources of amino acids for building proteins.  Discuss nitrogen fixation  The nitrogenase reaction  Families of amino acids based on their biosynthetic pathways and the relationship between amino acids and the citric acid cycle  Amino acid biosynthesis  Describe what is meant by the term essential amino acids. Describe the breakdown of dietary proteins Discuss the process of protein turnover Explain the process whereby an amino group is removed form an amino acid. Discuss the degradation of AA in muscle during prolonged exercise and fasting. Describe how amino acid degradation is linked to the TCA cycle. Discuss the Urea cycle e.g. ATP used, where performed, where the two N in urea are from. Explain what is meant by the terms keto- and glucogenic amino acids and provide examples. Give examples of conditions where there are errors in the degradation of amino acids.. 27 | CHEM206 @ ACU 27 Proteins are degraded into amino acids. Dietary proteins are a vital source of amino acids. Discarded cellular proteins are another source of amino acids. First step in protein degradation is the removal of the nitrogen Ammonium ion is converted to urea in most mammals. Carbon atoms are converted to other major metabolic intermediates. 28 | CHEM206 @ ACU 28 Nutrtion @ ACU 14  Amino Acid Digestion Protein Digestion and Absorption In order to be absorbed proteins must first be digested to smaller peptides and amino acids Different digestive enzymes target different motifs Digestive enzymes begin as zymogens to prevent unwanted digestion 29 | CHEM206 @ ACU 29 Summary of Enzymatic Digestion and Absorption Secretion Enzyme Substrate Action Final Product Saliva Ptyalin Starch Hydrolysis to form dextrins Gastric Pepsin Protein Hydrolysis juice of peptide bonds Gastric Fats Hydrolysis lipase into free fatty acids 30 | CHEM206 @ ACU 30 Nutrtion @ ACU 15  Summary of Enzymatic Digestion and Absorption —cont’d Secretion Enzyme Substrate Action Final Product Pancreatic Lipase Fat Hydrolysis to Fatty exocrine mono- acids secretion glycerides Cholesterol Cholesterol Hydrolysis to Choles- esterase esters of terol cholesterol and fatty acids α-Amylase Starch, Hydrolysis Dextrin, dextrins maltose 31 | CHEM206 @ ACU 31 Summary of Enzymatic Digestion and Absorption —cont’d Secretion Enzyme Substrate Action Final Product Pancreatic Trypsin Protein Hydrolysis Polypeptides exocrine secretion Chymotrypsin Protein Hydrolysis Polypeptides Carboxy- Polypep- Hydrolysis Amino acids peptidase tides Ribonuclease Ribonu- Hydrolysis Mono- cleic acids nucleotides Elastase Fibrous Hydrolysis Amino acids protein 32 | CHEM206 @ ACU 32 Nutrtion @ ACU 16  Summary of Enzymatic Digestion and Absorption —cont’d Secretion Enzyme Substrate Action Final Product Brush border Carboxy- Polypep- Hydrolysis Amino enzymes peptidase; tides acids aminopep- tidase; dipeptidase Entero- Trypsino- Activates to Polypep- kinase gen trypsin tidases and peptides Sucrase Sucrose Hydrolysis Glucose, fructose 33 | CHEM206 @ ACU 33 Summary of Enzymatic Digestion and Absorption —cont’d Secretion Enzyme Substrate Action Final Product Brush Isomaltase Dextrin Hydrolysis Glucose border enzymes Maltase Maltose Hydrolysis Glucose Lactase Lactose Hydrolysis Glucose, galactose Nucleotidases Nucleic acid Hydrolysis Nucleotides Nucleosidases Nucleosidases Hydrolysis Purine and and pyrimidine phosphorylase bases 34 | CHEM206 @ ACU 34 Nutrtion @ ACU 17  Cellular Protein Degradation Cellular proteins are degraded at different rates. The protein ubiquitin is used to mark cellular proteins for destruction. 35 | CHEM206 @ ACU 35 Ubiquitin Ubiquitin is activated and attached to proteins using a group of three enzymes E1 - Ubiquitin activating enzyme E2 - Ubiquitin-conjugating enyzme E3 - Ubiquitin-protein ligase 36 | CHEM206 @ ACU 36 Nutrtion @ ACU 18  Protein Turnover Protein Turnover There is no storage pool for protein Braz J Med Biol Res. 2012 Sep; 45(10): 875–890. 37 | CHEM206 @ ACU Published online 2012 Jun 8 37 Removal of Nitrogen The first step in amino acid catabolism is the removal of the nitrogen. The liver is the major site of protein degradation in mammals. Deamination produces α-keto acids, which are degraded to other metabolic intermediates. 38 | CHEM206 @ ACU 38 Nutrtion @ ACU 19  Conversion to Ammonium Ions α–Amino groups are converted to ammonium ions by the oxidative deamination of glutamate Reverse of this! 39 | CHEM206 @ ACU 39 Serine and Threonine The β–hydroxy amino acids, serine and threonine, can be directly deaminated 40 | CHEM206 @ ACU 40 Nutrtion @ ACU 20  Transporting Nitrogen to Liver Urea is produced in the Liver The alanine cycle is used to transport nitrogen to the liver 41 | CHEM206 @ ACU 41 Deamination In most terrestrial vertebrates the ammonium ion is converted to urea. 42 | CHEM206 @ ACU 42 Nutrtion @ ACU 21  The Urea Cycle The Urea Cycle Excess nitrogen is metabolised and secreted with three main endpoints Occurs almost exclusively in the liver and requires 4 ATP per molecule of urea 43 | CHEM206 @ ACU 43 The Urea Cycle 44 | CHEM206 @ ACU 44 Nutrtion @ ACU 22  Formation of Carbamoyl Phosphate Carbamoyl synthetase Free NH4 reacts with HCO3 to form carbamoyl phosophate. Reaction is driven by the hydrolysis of two molecules of ATP 45 | CHEM206 @ ACU 45 Formation of Citrulline Ornithine transcarbamoylase Citrulline is formed from transfer of the carbamoyl group to the γ-amino group of ornithine. 46 | CHEM206 @ ACU 46 Nutrtion @ ACU 23  Formation of Arginosuccinate Condensation of citrulline with aspartate to form arginosuccinate Two equivalent of ATP are required. 47 | CHEM206 @ ACU 47 Formation of Arginine and Fumarate Arginosuccinase Cleaves arginosuccinate to form arginine and fumarate 48 | CHEM206 @ ACU 48 Nutrtion @ ACU 24  Formation of Urea Arginase The arginine is hydrolyzed to produce the urea and to reform the ornithine. The ornithine reenters the mitochondrial matrix. 49 | CHEM206 @ ACU 49 Linked to Citric Acid Cycle The urea cycle is linked to the citric acid cycle: Kreb’s Bi-cycle!! 50 | CHEM206 @ ACU 50 Nutrtion @ ACU 25  Carbon Atoms The carbon atoms of degraded amino acids emerge as major metabolic intermediates. Degradation of the 20 amino acids funnel into 7 metabolic intermediates 51 | CHEM206 @ ACU 51 Glucogenic and ketogenic amino acids Glucogenic and ketogenic Amino acids broken down into a range of molecules such as pyruvate, acetyl- CoA and acetoacetyl-CoA Oxaloacetate is a key molecule Mammals cannot synthesise glucose from acetyl-CoA. Instead, they produce ketone bodies instead. 52 | CHEM206 @ ACU 52 Nutrtion @ ACU 26  53 | CHEM206 @ ACU 53 Amino Acid Metabolism Learning Objectives By the end of this topic you will understand :  List the sources of amino acids for building proteins.  Discuss nitrogen fixation  The nitrogenase reaction  Families of amino acids based on their biosynthetic pathways and the relationship between amino acids and the citric acid cycle  Amino acid biosynthesis  Describe what is meant by the term essential amino acids.  Describe the breakdown of dietary proteins  Discuss the process of protein turnover  Explain the process whereby an amino group is removed form an amino acid. Discuss the degradation of AA in muscle during prolonged exercise and fasting. Will cover later  Describe how amino acid degradation is linked to the TCA cycle.  Discuss the Urea cycle e.g. ATP used, where performed, where the two N in urea are from.  Explain what is meant by the terms keto- and glucogenic amino acids and provide examples.  Give examples of conditions where there are errors in the degradation of amino acids.. 54 | CHEM206 @ ACU 54 Nutrtion @ ACU 27