Lecture 10 Amino Acid Metabolism.pdf

Lecture 10 Amino Acid Metabolism Applied Biochemistry (NUR 112) Second Semester 2023-2024 Instructor: Dr. Roba Bdeir Dr. Roba Bdeir 1  Amino Acids: Disposal of Nitrogen Amino Acid (aa) are not stored by body  no protein exists...

Lecture 10 Amino Acid Metabolism Applied Biochemistry (NUR 112) Second Semester 2023-2024 Instructor: Dr. Roba Bdeir Dr. Roba Bdeir 1  Amino Acids: Disposal of Nitrogen Amino Acid (aa) are not stored by body  no protein exists to maintain a supply of aa for future use Thus, aa must be obtained from the diet, synthesized de novo, or produced from normal protein degradation 1st phase of catabolism involves the removal of α-amino gps forming ammonia & α-keto acid A portion of the free ammonia is excreted in the urine, but most is used in the synthesis of urea The carbon skeletons of the α-ketoacids can be metabolized to CO2 and water, glucose, fatty acids, or ketone bodies Structure of aa: – L-a-Amino acids are the structural or the building units of proteins – D-aa are present in diet, & efficiently metabolized by liver using D-aa oxidase (FAD-dependent enzyme) that catalyzes oxidative deamination of these isomers. The resulting α-ketoacids can enter the general pathways of amino acid metabolism, and be reaminated to L-isomers, or catabalized for energy. 2 Abbreviations for the 20 Amino Acids Hydrophilic Hydrophobic 3  Metabolic Classification of Amino Acids Amino acids can be classified as glucogenic, ketogenic, or both: Glucogenic aa are whose catabolism yields: – pyruvate or – one of the intermediates of the citric acid cycle ketogenic aa whose catabolism yields either: – acetoacetate – one of its precursors (acetyl CoA or acetoacetyl CoA) are termed 4  Amino acid metabolism Amino acid pool: – aa pool is small (~ 90–100 g of aa) in comparison with amount of body’s protein (~12 kg in 70-kg man) – It is conceptually at the center of whole-body nitrogen metabolism. – The aa pool is said to be in a steady state (input=output) Protein turnover: – Proteins are constantly degraded & synthesized which is regulated by concentration of protein in cell – 300-400g proteins are hydrolyzed & resynthesized/day – Protein turnover varies: short lived (regulatory & misfolded proteins), long-lived (most of tissue proteins) & structurally stable (collagen) 5  Nitrogen Balance Nitrogen balance occurs when the amount of nitrogen consumed equals that of the nitrogen excreted in the urine, sweat, and feces +ve Nitrogen Balance = N2 intake > N2 output new tissues are build up: 1. During growth (growing children). 2. Pregnancy. 3. Muscular training. Prolonged periods of -ve nitrogen 4. Convulsions from different diseases. balance may lead to death. -ve Nitrogen Balance = N2 Output is > N2 intake 1. ↓ protein intake: e.g. starvation, malnutrition & G.I.T. diseases. 2.  Loss of proteins: e.g. in chronic hemorrhage, albuminuria & lactation on an inadequate protein diet. 3. Increased of protein catabolism: e.g. fever, hyperthyroidism, DM, Cushing syndrome, advanced cancer & post-surgical. 6  Protein Metabolism Protein degradation occurs by 2 enzyme systems: 1) energy dependent ubiquitin-protesome mechanism (endogenous proteins) 2) non-energy dependent lysosomes (extracellular protein) Oxidized or ubiquitin tagged proteins are preferentially degraded Chemical signals for protein degradation (t1/2 of a protein is influenced by nature of N-terminal residue) – Serine (S) at N-terminal: long t1/2 (>20 hr) – Aspartate (D) at N-terminal: short t1/2 (3 min) – Proteins rich in the sequence (PEST) are rapidly degraded (i.e. proline, glutamate, serine, & threonine) 7  Digestion of proteins protein is antigenic i.e. able to stimulate an immunologic response. The digestion of protein destroys its antigenecity. 1) In the stomach: a. gastric acid: denature the protein b. Pepsin (major proteolytic enzyme in stomach) : – produced & secreted by chief cells of stomach as inactive zymogen, pepsinogen, which activated by HCl produced by parietal cells of stomach. – Pepsin catalyzes the cleavage of proteins into smaller polypeptides 2) In small intestine: – large polypeptides are cleaved to oligopeptides & aa by a gp of pancreatic proteases; each has a different specificity (trypsin cleaves only at C-terminal of arginine or lysine). – Enteropeptidase converts the pancreatic trypsinogen to trypsin which starts a cascade of proteolytic activity, because trypsin is the activator of all the pancreatic zymogens 8  Abnormalities in protein digestion In individuals with a deficiency in pancreatic secretion (chronic pancreatitis, cystic fibrosis, or surgical removal of the pancreas)  digestion & absorption of fat & protein is incomplete  abnormal appearance of lipids (Steatorrhea) & undigested protein in feces Digestion of oligopeptides by enzymes of the small intestine – Luminal surface of intestine contains aminopeptidase (an exopeptidase that repeatedly cleaves N-terminal residue of oligopeptides to produce free aa & smaller peptides). Absorption of amino acids and dipeptides – Free aa & dipeptides are taken up by the intestinal epithelial cells. – dipeptides are hydrolyzed in cytosol to aa before being released into the portal system (only free amino acids are found in the portal vein) – The absorption of amino acid is active process that needs energy (ATP). 9  Transport of Amino Acids into Cells At least seven different transport systems are known that have overlapping specificities for different aa. Small intestine & proximal tubule of kidney have common transport systems for aa uptake  one system is responsible for uptake of cystine & dibasic aa, ornithine, arginine, & lysine. Cystinuria is the most common genetic error of aa transport: – This carrier system is defective, & all four amino acids appear in urine – The disease expresses itself clinically by the precipitation of cystine to form kidney stones (calculi) that may block the urinary tract. – Oral hydration is important in treatment for this disorder 10  Removal of nitrogen from aa Removing α-amino gp is essential for producing energy from aa transamination & oxidative deamination rxns provide ammonia & aspartate (sources of urea nitrogen) 1st transfer their α-amino gp to α-ketoglutarate to produce an α- ketoacid & glutamate (transamination) Glutamate can be oxidatively deaminated or used in synthesis of nonessential aa. Transamination The transfer of amino groups from one carbon skeleton to another is catalyzed by a family of enzymes called aminotransferases  found in cytosol of body cells (especially liver, kidney, intestine, & muscle) All aa (except lysine & threonine) participate in transamination at some point in their catabolism Lysine and threonine lose their α-amino groups by deamination 11  Aminotransferases Each aminotransferase is specific for one or, few amino group donors & named after that enzyme For example: Alanine aminotransferase (ALT): catalyzes (reversibly) transfer of amino group of alanine to α-ketoglutarate  formation of pyruvate & glutamate. Aspartate aminotransferase (AST): transfers amino groups from glutamate to oxaloacetate, forming aspartate, which is used as a source of nitrogen in the urea cycle Both reaction are reversible 12  Mechanism of action of aminotransferases All aminotransferases require the coenzyme pyridoxal phosphate to transfer amino gp of aa to it & generate pyridoxamine-P, which then reacts with an α-keto acid to form aa, at the same time regenerating the original aldehyde form of the coenzyme 13  Diagnostic value of plasma aminotransferases Aminotransferases are normally intracellular enzymes (low levels in plasma) The presence of elevated plasma levels of aminotransferases indicates damage to cells rich in these enzymes. Two aminotransferases (AST & ALT) are of particular diagnostic value when they are found in plasma. a. hepatic disease: Plasma AST & ALT are elevated in nearly all liver diseases, specially in extensive cell necrosis (severe viral hepatitis, toxic injury, & prolonged circulatory collapse). Elevated serum bilirubin results from hepatocellular damage that decreases the hepatic conjugation & excretion of bilirubin b. Nonhepatic disease: Aminotransferases may be elevated in nonhepatic disease (myocardial infarction & muscle disorders) but those can be clinically distinguished. 14  Glutamate dehydrogenase (the oxidative deamination of aa) Transfer amino gps from glutamate, oxidative deamination, by glutamate dehydrogenase results in liberation of amino group as free ammonia  occurs primarily in liver & kidney. Glutamate  only aa undergoes rapid oxidative deamination Glutamate dehydrogenase can use either NAD or NADP as a coenzyme. NAD is used primarily in oxidative deamination & NADPH is used in reductive amination Rxn can be used to synthesize aa from corresponding α-ketoacids Regulation: Direction of rxn depends on relative concentrations of glutamate, α- ketoglutarate & ammonia, & ratio of oxidized to reduced coenzymes. After ingestion of a meal containing protein, liver glutamate levels  & enhance aa degradation & formation of ammonia ATP & GTP are allosteric inhibitors of glutamate dehydrogenase, whereas ADP & GDP are activators. 15  Transport of ammonia from tissues to liver There are two mechanisms: 1) Found in most tissues, uses glutamine synthetase to combine ammonia with glutamate to form glutamine (a nontoxic transport form of ammonia) – Glutamine is transported in blood to liver where is cleaved by glutaminase to produce glutamate & free ammonia 2) Used primarily by muscle, involves transamination of pyruvate (alanine aminotransferase) to form alanine – Alanine is transported by blood to liver, where it is converted to pyruvate by transamination (pyruvate is used in gluconeogenesis)  called glucose-alanine cycle. 16  UREA CYCLE Urea is the major disposal form of amino groups derived from aa (90% of nitrogen-containing components of urine). One nitrogen of urea molecule is supplied by free NH3, & the other nitrogen by aspartate, the C & O of urea are derived from CO2 17  UREA CYCLE Urea is produced by the liver, and then is transported in the blood to the kidneys for excretion in the urine. Reactions of the cycle: 1) Formation of carbamoyl phosphate by carbamoyl phosphate synthetase I which requires 2 ATP. N-acetylglutamate is required as allosteric activator. 18  UREA CYCLE 2. Formation of citrulline: Ornithine & citrulline are basic aa that participate in urea cycle (But not into cellular proteins, no codons). citrulline is transported to the cytosol. 3. Citrulline condenses with aspartate to form argininosuccinate. α-amino gp of aspartate provides the 2nd nitrogen that is ultimately incorporated into urea, which is driven by the cleavage of ATP to AMP & Ppi. 19 4. Argininosuccinate is cleaved to yield arginine & fumarate. The arginine formed by this reaction serves as the immediate precursor of urea. Fumarate can reenter the TCA cycle 5. Cleavage of arginine to ornithine & urea by arginase occurs almost exclusively in the liver, whereas other tissues (kidney), can synthesize arginine by these reactions. 6. Fate of urea: Urea diffuses from the liver, and is transported in the blood to the kidneys, where it is filtered and excreted in the urine. 20  UREA CYCLE A portion of the urea diffuses from the blood into the intestine, & is cleaved to CO2 & NH3 by bacterial urease. This ammonia is partly lost in the feces and is partly reabsorbed into the blood. In patients with kidney failure, plasma urea levels are elevated (hyperammonemia), promoting a greater transfer of urea from blood into the gut. Oral administration of neomycin reduces the number of intestinal bacteria responsible for this NH3 production. 21  Overall stoichiometry of urea cycle synthesis of urea is irreversible, with a large, negative ΔG. Regulation of the urea cycle N-Acetylglutamate is an essential activator for carbamoyl phosphate synthetase I (rate- limiting step) N-Acetylglutamate is synthesized from acetyl coenzyme A and glutamate by N- acetylglutamate synthase, in a reaction for which arginine is an activator. the intrahepatic concentration of N-acetylglutamate increases after ingestion of a protein- rich meal, which provides both the substrate (glutamate) and the regulator of N- acetylglutamate synthesis. This leads to an increased rate of urea synthesis. 22  Metabolism of ammonia Slight increase in the concentration of urea in blood leads to hyperammonemia which is toxic to the CNS Sources of ammonia: 1. From aa: mainly in liver by the aminotransferase and glutamate dehydrogenase reactions 2. From glutamine: The kidneys form ammonia from glutamine by the action of renal glutaminase. Ammonia is also obtained from the hydrolysis of glutamine by intestinal glutaminase. 3. From bacterial action in the intestine: Ammonia is formed from urea by the action of bacterial urease in the lumen of the intestine. 4. From amines: Amines obtained from the diet, and monoamines that serve as hormones or neurotransmitters 5. From the catabolism of purines and pyrimidines: amino groups attached to the rings are released as ammonia. 23  Transport of ammonia in circulation As urea: – the most disposal form of ammonia which moves from liver to the kidney As Glutamine: – Occurs primarily in the muscle and liver and nervous system. – Circulating glutamine is removed by liver & kidneys & deaminated by glutaminase. Hyperammonemia – when the liver function is compromised, due either to genetic defects of the urea cycle, or liver disease, blood levels can rise above 1000 µmol/L. – hyperammonemia is a medical emergency, because ammonia has a direct neurotoxic effect on the CNS (tremors, slurring of speech, somnolence, vomiting, cerebral edema, and blurring of vision). – At high concentrations, ammonia can cause coma and death. 24  Hyperammonemia Acquired hyperammonemia: – It may be due to viral hepatitis, ischemia, or hepatotoxins. – Cirrhosis of liver caused by alcoholism, hepatitis, or biliary obstruction result in formation of collateral circulation around liver. Hereditary hyperammonemia: – Ornithine transcarbamoylase deficiency, which is X-Iinked, is the most common of these disorders, affecting males predominantly – Other urea cycle disorders are autosomal recessive disorders. Failure to synthesize urea leads to hyperammonemia during 1 st weeks following birth leading to mental retardation Treatment: ü limiting protein in the diet ü administering compounds that bind covalently to aa, producing nitrogen-containing molecules that are excreted in urine (phenylbutyrate given orally is converted to phenylacetate that condenses with glutamine to form phenylacetylglutamine & excreted) 25