Amino Acid Metabolism (Week One Slides) PDF

9/16/24 LECTURER: Dr Ofentse Pooe AMINO ACID METABOLISM 1 MAJOR CONCEPTS TO BE UNDERSTOOD 1. Dietary proteins are the primary source of biologically useful nitrogen in our bodies. Other sources are internal protein breakdown and synthesis using carbon skeletons from o...

9/16/24 LECTURER: Dr Ofentse Pooe AMINO ACID METABOLISM 1 MAJOR CONCEPTS TO BE UNDERSTOOD 1. Dietary proteins are the primary source of biologically useful nitrogen in our bodies. Other sources are internal protein breakdown and synthesis using carbon skeletons from other amino acids or carbohydrates 2. The general scheme for the further metabolism of "digested" amino acids involves the transfer of the amino group to alpha-ketoglutarate forming glutamate plus an alpha-keto acid. 3. The glutamate produced is transported to liver mitochondria and deaminated by glutamate dehydrogenase. 4. Glutamine and alanine transport ammonia formed in other tissues to the liver. 5. Nitrogen is excreted as ammonia or urea. High serum levels of ammonia could indicate liver disease. 2 1  9/16/24 INTRODUCTION Amino acids are distinguished by the fact that in addition to C, H and O, they also have N as amino nitrogen in their structures Some species cannot synthesize the carbon skeletons of every amino acid The pool of amino acids in the body represents contributions from three main sources :diet; internal protein breakdown; and synthesis using carbon skeletons from other amino acids or carbohydrate 3 4 2  9/16/24 INTRODUCTION Some species cannot synthesize the carbon skeletons of every amino acid Mammals for example can make only about half of the amino acids they require, the rest called essential amino acids must be obtained from the diet Non-essential amino acids are those that mammals can synthesize in sufficient quantity, provided they receive adequate total dietary protein 5 Methionine and phenylalanine are respectively precursors of cysteine and tyrosine The latter amino acids can become essential if adequate amounts of the former are not supplied in the diet Arginine is a conditionally essential amino acid. Although synthesized in the body, the amounts are not adequate during growth 6 3  9/16/24 7 AMINO ACID POOL 8 4  9/16/24 PROTEIN TURNOVER Most proteins in the body are constantly being synthesized and then degraded, permitting the removal of abnormal or unneeded proteins. For many proteins, regulation of synthesis determines the concentration of protein in the cell, with protein degradation assuming a minor role. For other proteins, the rate of synthesis is constitutive (that is, essentially constant), and cellular levels of the protein are controlled by selective degradation. 1. Rate of turnover: In healthy adults, the total amount of protein in the body remains constant because the rate of protein synthesis is just sufficient to replace the protein that is degraded. This process, called protein turnover, leads to the hydrolysis and resynthesis of 300–400 g of body protein each day. The rate of protein turnover varies widely for individual proteins. Short-lived proteins (for example, many regulatory proteins and misfolded proteins) are rapidly degraded, having half-lives measured in minutes or hours. Long-lived proteins, with half-lives of days to weeks, constitute the majority of proteins in the cell. Structural proteins, such as collagen, are metabolically stable and have half-lives measured in months or years. 9 AMINO ACIDS FROM BODY PROTEINS The ubiquitin (Ub)-proteasome pathway Amino acids can be derived from degradation of (UPP) of protein degradation. proteins in the body either because they are defective or as part of normal regulatory processes. There are two main routes by which they are hydrolysed to individual amino acids: i) by lysosomal cathepsins which are proteases with acid pH optima, and ii) by the cytosolic 2000 kD, 26S proteasome in an ATP-dependent process after the proteins are tagged with ubiquitin Ubiquitin is a 76-residue monomeric protein that marks a protein for degradation by proteasome once it tags the protein via a covalent linkage to a lysine residue on the protein Stewart H. Lecker et al. JASN 2006;17:1807-1819 ©2006 by American Society of Nephrology 10 5  9/16/24 PROTEIN DEGRADATION 2. Protein degradation: There are two major enzyme systems responsible for degrading proteins: the adenosine triphosphate (ATP)-dependent ubiquitin-proteasome system of the cytosol, and the ATP- independent degradative enzyme system of the lysosomes. Proteasomes selectively degrade damaged or short-lived proteins. Lysosomes use acid hydrolases to nonselective degrade intracellular proteins (“autophagy”) and extracellular proteins (“heterophagy”), such as plasma proteins, that are taken into the cell by endocytosis. a. Ubiquitin–proteasome proteolytic pathway: Proteins selected for degradation by the cytosolic ubiquitin-proteasome system are first modified by the covalent attachment of ubiquitin (Ub), a small, globular, nonenzymic protein that is highly conserved across eukaryotic species. Ubiquitination of the target substrate occurs through isopeptide linkage of the α-carboxyl group of the C- terminal glycine of Ub to the ε-amino group of a lysine on the protein substrate by a three-step, enzyme- catalyzed, ATP-dependent process. Proteins tagged with Ub are recognized by a large, barrel-shaped, macromolecular, proteolytic complex called a proteasome. The proteasome unfolds, deubiquitinates, and cuts the target protein into fragments that are then further degraded by cytosolic proteases to amino acids, which enter the amino acid pool. Ub is recycled. It is noteworthy that the selective degradation of proteins by the ubiquitin-proteosome complex (unlike simple hydrolysis by proteolytic enzymes) requires energy in the form of ATP. The ubiquitin-proteasome degradation pathway of proteins. 11 PROTEIN DEGRADATION b. Chemical signals for protein degradation: Because proteins have different half-lives, it is clear that protein degradation cannot be random but, rather, is influenced by some structural aspect of the protein. For example, some proteins that have been chemically altered by oxidation or tagged with ubiquitin are preferentially degraded. The half-life of a protein is also influenced by the amino (N)-terminal residue. For example, proteins that have serine as the N-terminal amino acid are long-lived, with a half-life of more than 20 hours, whereas those with aspartate at their N-terminus have a half-life of only 3 minutes. Additionally, proteins rich in sequences containing proline, glutamate, serine, and threonine (called PEST sequences after the one-letter designations for these amino acids) are rapidly degraded and, therefore, have short half-lives. The ubiquitin-proteasome degradation pathway of proteins. 12 6  9/16/24 13 14 7  9/16/24 Dietary amino acids Dietary protein is hydrolysed by proteolytic enzymes in the gastrointestinal tract (GIT) e.g. pepsin (stomach), trypsin and chymotrypsin (from pancreatic juice), peptidases (released as zymogens by the pancreas) e.g. carboxypeptidases A and B The main products are individual amino acids 15 16 8  9/16/24 Dietary amino acids 17 ABSORPTION OF AMINO ACIDS In the small intestine amino acids are absorbed from the lumen through Na+ -dependent transport, facilitated diffusion and the γ-glutamyl cycle. On the serosal side of the intestinal mucosa they are transported into the blood by facilitated diffusion using different carriers In the γ-glutamyl cycle, the amino acid reacts with glutathione (γ-glutamyl-cysteinyl-glycine) in a reaction catalysed by γ-glutamyl transpeptidase. Following release of the amino acid inside the mucosal cell, glutathione is regenerated 18 9  9/16/24 19 Amino acid synthesis 20 10  9/16/24 21 22 11  9/16/24 Methionine and phenylalanine are respectively precursors of cysteine and tyrosine The latter amino acids can become essential if adequate amounts of the former are not supplied in the diet Arginine is a conditionally essential amino acid. Although synthesized in the body, the amounts are not adequate during growth 23 In the small intestine amino acids are absorbed from the lumen through Na+ -dependent transport, facilitated diffusion and the γ-glutamyl cycle. On the serosal side of the intestinal mucosa they are transported into the blood by facilitated diffusion using different carriers In the γ-glutamyl cycle, the amino acid reacts with glutathione (γ-glutamyl-cysteinyl-glycine) in a reaction catalysed by γ-glutamyl transpeptidase. Following release of the amino acid inside the mucosal cell, glutathione is regenerated 24 12  9/16/24 25 Amino Acid Metabolism in the Liver 27 13  9/16/24 Amino Acid Metabolism During Periods of Fasting 28 Route of synthesis of amino acids 29 14  9/16/24 In this chapter we are going to consider the metabolism of the 20 common amino acids from two points of view: The origins and fates of their carbon skeletons 30 Definitions to remember 1. Transamination is the transfer of an amine group from an amino acid to a keto acid (amino acid without an amine group), thus creating a new amino acid and keto acid 2. Deamination is the removal of the amine group as ammonia (NH3) 31 15  9/16/24 The major issue with deamination is that high levels of ammonia are toxic, leading to hyperammonemia. 32 Assimilation of Ammonia Ammonia is assimilated into a large number of low molecular weight metabolites, often via the amino acids glutamate and glutamine At physiological pH the main ionic form of ammonia is the ammonium ion, NH4+ The reductive amination of α-ketoglutarate to glutamate by glutamate dehydrogenase is one highly efficient route for the incorporation of ammonia into the central pathways of amino acid metabolism 33 16  9/16/24 34 Glutamate dehydrogenase in mammals Mammals probably assimilate very little nitrogen as free ammonia because they get most of their nitrogen from amino acids and nucleotides in the diet The primary role of glutamate dehydrogenase in mammals is the degradation of amino acids and the release of NH4+ Another reaction critical to the assimilation of ammonia in many organisms is the formation of glutamine from glutamate and ammonia This reaction is catalysed by glutamine synthetase 35 17  9/16/24 Ammonia incorporated into glutamate and glutamine The glutamate hydrogenases of some species or tissues are specific for NADH while others are specific for NADPH. Some can use either. 36 Transamination reactions Transamination is the transfer of an amino group from one molecule to another Most amino acids are deaminated by transamination, a chemical reaction that transfers an amino acid to a ketoacid to form a new amino acid The amino group of glutamate can be transferred to many α-keto acids in reactions catalysed by enzymes known as transaminases or aminotransferases 37 18  9/16/24 glutamate α-ketoglutarate 38 Transamination reactions All known transamination reactions require the enzyme pyridoxal phosphate (further reading section 7.8 of your recommended textbook) The transaminases catalyse near-equilibrium reactions The direction in which the reactions proceed in vivo (flux) depends on the supply of substrates and the removal of products 39 19  9/16/24 41 REMOVAL AND DISPOSAL OF AMINO ACID NITROGEN There are other reactions that also produce ammonia in the body and these include: i) The hydrolysis of side-chain amides of asparagine and glutamine by asparaginase and glutaminase which yield aspartate and glutamate, respectively ii) Pyridoxal-5-phosphate dependent dehydration of serine and threonine catalysed by serine (threonine) dehydratase which yields pyruvate and α- ketobutyrate, respectively, and ammonia iii) Deamination of histidine by histidinase (histidine ammonia lyase) which yields ammonia and urocanate. iv) The purine nucleotide cycle which operates in muscle (Fig 6) v) L and D amino oxidase reactions. The D amino acid oxidase is mainly found in the kidney. The function of this enzyme maybe to racemize D- amino acids to L-amino acids which are the form used by enzyme systems in the body. The reaction produces α-keto acid (Fig 7) then the amino group is added in L-configuration by L-aminotransferase in the body. The sources of amino acids can be the diet or gut bacteria. 42 20  9/16/24 Routes for disposal of the nitrogen-containing waste products of amino acid metabolism in various animals Excess nitrogen is excreted in: 1) aquatic animals → ammonia 2) Birds and most reptiles → uric acid 43 44 21  9/16/24 The Nitrogen Cycle and Nitrogen Fixation The nitrogen needed for amino acids (and for the heterocyclic bases of nucleotides) comes from two major sources: 1) Nitrogen gas in the atmosphere 2) Nitrates (NO3−) in soil and water 45 The Nitrogen Cycle and Nitrogen Fixation Atmospheric N2 , which constitutes about 80% of the atmosphere, is the ultimate source of biological nitrogen This molecule can be metabolised, or fixed, by only a few species of bacteria N2 and NO3− must be reduced to ammonia in order to be used in metabolism The ammonia produced is then incorporated into amino acids via glutamate, glutamine, and carbamoyl phosphate 46 22  9/16/24 The Nitrogen Cycle and Nitrogen Fixation N2 is chemically unreactive - because of the strength of the great strength of the N≡ N triple bond Some bacteria have a very specific, sophisticated enzyme called nitrogenase , that can catalyse the reduction of N2 to ammonia in a process called nitrogen fixation 47 The Nitrogen Cycle and Nitrogen Fixation There are two additional nitrogen-converting processes in addition to biological nitrogen fixation During lightning storms , high voltage discharges catalyse the oxidation of N2 to nitrate (NO3−) and nitrite (NO2−) Nitrogen is converted to ammonia for use in plant fertilizers by an energetically expensive industrial process that requires high temperature and pressure as well as special catalysts to drive the reduction of N2 by H2 48 23  9/16/24 The Nitrogen Cycle and Nitrogen Fixation The flow of nitrogen from N2 to nitrogen oxides , ammonia , and nitrogenous biomolecules and then back to N2 is called the nitrogen cycle Most of the nitrogen shuttles between ammonia and nitrate Ammonia from decayed organisms is oxidized by soil bacteria to nitrate This formation of nitrate is called nitrification Some anaerobic bacteria can reduce nitrate to nitrite to N2 (denitrification) 49 Ammonia is essential for life and bacteria are the only organisms capable of producing it from atmospheric nitrogen Half of all biological nitrogen fixation is performed by various species of cyanobacteria in the ocean The other half comes from soil bacteria Blooms of Trichodesmium - one of the main nitrogen-fixing species of cyanobacteria https://www.researchgate.net/profile/NV_Madhu/publication/27667323/figure/fig3/AS:277169134686212@1443093679915/Figure-6-Trichodesmium-erythraeum-bloom.png 50 24