BIOCHEMISTRY TRANS 7a - Nitrogen Disposal.pdf

1A BIOCHEMISTRY AMINO ACIDS: NITROGEN DISPOSAL (Part 1) DR. B. JANDOC PART 1 OVERVIEW Amino Ac...

1A BIOCHEMISTRY AMINO ACIDS: NITROGEN DISPOSAL (Part 1) DR. B. JANDOC PART 1 OVERVIEW Amino Acids  Contain CHON atoms  Cannot be stored, excess are immediately degraded  obtained from Diet, De Novo Synthesis, and Normal Protein Degradation  Catabolism 1. 1st Phase - Removal of alpha-Amino Groups through Transamination and Oxidative Deamination - Products include Ammonia and Corresponding alpha-Ketoacids - Ammonia is most used in urea synthesis and is excreted in urine - alpha-Ketoacids are carbon skeletons of amino acids Gastrointestinal Input 2. 2nd Phase - Digested and Absorbed Daily include - carbon skeletons of alpha-ketoacids are Dietary Protein (70-100 gm) and common intermediates of energy-producing Endogenous Protein (35-200 gm) metabolic pathways - Polypeptides are hydrolyzed to free amino acids before absorption - Proteolytic Enzymes are involve in OVERALL NITROGEN METABOLISM proteolysis of dietary protein A. Amino Acid Pool  free amino acids distributed throughout the body  amino acids released by hydrolysis of dietary or tissue protein  essential amino acids are carbon skeletons that cannot be synthesized by the organism - histidine - lysine - threonine Endogenous Protein Proteolysis - isoleucine - body proteins are continuously broken down - methionine to free amino acids - tryptophan - degradation rate vary for individual proteins - leucine Liver Enzymes - few-hour half-lives - phenylalanine RBCs (Hemoglobin) - 120 day life span - valine Structural Proteins (Collagen) - too long to - arginine measure  Inputs - Factors Affecting Protein Degradation 1. Dietary Protein Rates: 2. Proteolysis of Cellular Proteins 1. Protein Denaturation - adults degrade 1-2% of their total body protein - loss of its preferred native (mainly muscle protein) configuration - of the amino acids liberated, 75-80% is - accelerates proteolytic breakdown recaptured which is used in the biosynthesis of 2. Lysosomal Activation new tissue protein and the other 20-25% forms - increases intracellular proteolytic rate urea 3. Glucocorticoids - carbon skeletons degraded to amphibolic - increase muscle tissue protein intermediates degradation Trans Finals 7a | ABACCO, ALDERITE, ASISTIN, BALANZA, BAYAS, BIANG 1 of 5  1A BIOCHEMISTRY AMINO ACIDS: NITROGEN DISPOSAL (Part 1) DR. B. JANDOC B. Protein Turnover  During Normal Conditions: 4. Thyroid Hormone Excess rate of protein synthesis = rate of protein degradation - increase protein turnover  Three Functions: 5. Insulin 1. Proteins serve as a form of long-term energy storage that - reduce proteolysis is utilized to provide gluconeogenic precursors and ketone - increase protein synthesis bodies - Factors Affecting Protein Stability: 2. Degradation eliminates abnormal and damaged proteins o Abnormal, Defective, and (accumulation is potentially hazardous to cell function) Damaged Proteins have no 3. The regulation of metabolic activity under changing use in the body and may physiological conditions inhibit processes that require requires the degradation of one set of regulatory proteins the functional protein that is and its replacement by a new set why they must be removed  Rate of Turnover o Inducible Enzymes must be a. Rapidly Degraded removed when their activities  Proteins Functioning Outside the Cell are no longer required or - half-lives of hours to days beneficial ex. Digestive Enzymes, Plasma o Stability of Cytosolic Proteins Proteins and Their Reactivity  Short-Lived Proteins Towards Ubiquitin must be - half-lives of minutes to hours correlated with the nature of ex. Many Regulatory Proteins, the amino acid terminal of the Misfolded Proteins protein b. Long-Lived Proteins - Muscle Protein Breakdown Estimation - half-lives of days to weeks some histidine residues of muscle protein ex. majority of proteins in the cell complex actomyosin -> methylated after c. Structural Proteins their incorporation -> actomyosin breakage - metabolically stable -> 3-methylhistidine liberation -> excreted - half-lives of months to years in the urine ex. Collagen Protein Degradation De Novo Synthesis a. Major Enzyme Systems for Degrading Damaged or Unneeded Proteins  Outputs  Ubiquitin-Proteasome Mechanism - Protein Synthesis as major drain of the pool o energy-dependent - Amino Acid Catabolism to urea and CO2 o Ubiquitin (amino acid degradation is reduced in  important role in designating starvation but is never turned off) proteins to be degraded - Synthesis of Special Compounds  Ubiquitination of Target Protein Destined for  Excess Amino Acids Degradation : linkage of - any amino acid not immediately used must lysine alpha-amino group of be converted to glycogen or fat stores proteins to carboxyl glycine of ubiquitin  Protein Content of the Body  Three Enzymes Required in - total amount of protein in the body = 12 kg Ubiquitination : in a 70 kg individual 1. Protein E1 (Ubiquitin- o Extracellular Activating Enzyme) forms a - half of endogenous proteins (6-7 kg) associated thioester bond with ubiquitin with skeleton and other supporting tissues 2. Protein E2 (Ubiquitin- - Collagen is the major protein component Conjugating Enzyme) receives - Plasma Proteins are highly dynamic (normal ubiquitin molecule from ubiquitin- half-life of plasma albumin = 20 days) E1 complex forming another o Intracellular thioester bond and transfers it to - other half of body protein E3 - more dynamic with continual protein 3. Protein E3 (Ubiquitin-Protein synthesis and degradation Ligase) transfers activated ubiquitin to the Lys ε-amino group of a previously selected target protein Trans Finals 7a | ABACCO, ALDERITE, ASISTIN, BALANZA, BAYAS, BIANG 2 of 5  1A BIOCHEMISTRY AMINO ACIDS: NITROGEN DISPOSAL (Part 1) DR. B. JANDOC B. Protein Digestion by Pancreatic Enzymes o 26S Proteasomes  Proteins->stomach pepsin->large polypeptides->small  degrade endogenous intestines->pancreatic proteases->oligopeptides + amino proteins acids o 19S Cap  Release of Zymogens  recognizes ubiquitinated  Cholecystokinin-Pancreozymin and Secretin proteins and unfolds - influence pancreatic acinar cells them for degradation  Cholecystokinin  Lysosomal Degradative Enzymes - secretion stimulated by liberated peptides in o degrade extracellular proteins the duodenum - sets stage for pancreatic proteolysis Signals for Protein Turnover  Trypsinogen a. Preferentially Degraded - activated by enteropeptidase by cleaving off - proteins chemically altered by oxidation 6 amino acids b. PEST Sequences Proteins  Activation of Zymogens - proteins with segments rich in Pro (P), Glu (E),  Enteropeptidase (Enterokinase) Ser (S), and Thr (T) [called PEST sequences] - remove hexapeptide from the NH2-terminus c. Half-Life of a Protein of trypsinogen resulting to trypsin - influenced by the nature of the N-terminal  Trypsin residue (N-end rule) - Activates Other Trypsinogen Molecules and  Protein with serine as N-terminal amino All Other Pancreatic Zymogens acid : half-life of >20 hours - Pancreatic Zymogens include Endopeptidase  Protein with aspartate as N-terminal Proenzymes (Chymotrypsin, Elastase) & amino acid : half-life of about 3 Exopeptidase Proenzymes minutes (Carboxypeptidase A, Carboxypeptidase B)  Abnormalities in Protein Digestion C. Role of Dietary Protein in Overall Nitrogen Metabolism  chronic pancreatitis, cystic fibrosis,  Provision of Energy pancreatectomy ->pancreatic secretion deficiency  Primarily (carbohydrates, triacylglycerols) ->incomplete digestion and absorption of fat and  Secondarily (proteins) proteins->steatorrhea and undigested protein in  Recommended Dietary Allowance for Protein the feces  RDA - 56 gm protein/day for a 70 kg individual  Consequences of Diets Low in Protein C. Oligopeptide Digestion by Enzymes of the Small Intestines  Kwashiorkor syndrome  Aminopeptidases and Dipeptidases  Consequences of Diets High in Protein  at the luminal surface of the intestines  Excess Amino Acids are converted to glucose and fats  repeatedly cleaves the N-terminal residue from  Amino Groups are converted to ammonia oligopeptides to free amino acids and smaller peptides  Dipeptides and Tripeptides DIGESTION of DIETARY PROTEINS  absorbed and digested to free amino acids within intestinal epithelial cells - proteins are too large to be absorbed by the intestines that is why D. Amino Acid and Dipeptide Absorption they are hydrolyzed first to amino acids  free amino acids and dipeptides->intestinal epithelial cells- - Proteolytic Enzymes are produced by stomach, pancreas and small >dipeptide hydrolysis in the cytosol->free amino acids- intestines >portal system ->liver A. Protein Digestion by Gastric Secretions  Gastric juice AMINO ACID TRANSPORT into CELLS  Stomach Acid (Hydrochloric Acid) - too dilute (pH 2-3) to hydrolyze proteins A. Active Transport Systems (7) - provides acid environment for pepsin  driven by ATP hydrolysis, movement of amino acids from - denature proteins, extracellular (lower concentration) to intracellular (higher  Pepsin concentration) compartment - acid-stable endopeptidase  size of gradient vary with different amino acids (highest for - Stomach Serous Cells secrete pepsinogen glutamate and glutamine) - Major Products of Peptic Hydrolysis of  COAL System is responsible for the uptake of Cystine and Proteins include Large Polypeptides and Dibasic Amino Acids like ornithine, arginine and lysine Some Free Amino Acids  Luminal Transport is Na+-dependent  Contraluminal Transport is Na+-independent Trans Finals 7a | ABACCO, ALDERITE, ASISTIN, BALANZA, BAYAS, BIANG 3 of 5  1A BIOCHEMISTRY AMINO ACIDS: NITROGEN DISPOSAL (Part 1) DR. B. JANDOC B. Disorders Associated with Amino Acid Transport Defects  leads to elevated levels of specific amino acids in the urine (amino acidurias)  catalyzed by aminotransferases (transaminases)  amino acids absorbed as peptides in the intestines by  found in the cytosol and mitochondria of cells benign or cause throughout the body only minor health problems  all amino acids except lysine and threonine (lose their α-amino groups by deamination) participate in transamination at some point in their catabolism  Products include α-Ketoacid & Glutamate  Substrate Specificity of Aminotransferases is named after the specific amino group donor; amino group acceptor is almost always alpha- ketoglutarate  ALT (Alanine Aminotransferase)/GPT  Hartnup’s Disease (Glutamate : Pyruvate Transaminase)  rare autosomal recessive defect in the intestinal which transfers amino groups from alanine and renal transporters to alpha-ketoglutarate  transport system defect for large neutral and  AST (Aspartate Aminotransferase)/GOT aromatic amino acids (Glutamate : Oxaloacetate Transaminase)  due to a loss of tryptophan (nicotinamide transfers amino groups from glutamate to precursor) oxaloacetate  many aspects of the presentation mimic niacin  Mechanism of Action of Aminotransferases (vitamin B3) deficiency (pellagra)  Pyridoxal Phosphate which is a derivative of  exhibit pellagra-like skin lesions and neurologic vitamin B6 is covalently linked to the α- manifestations ranging from ataxia to frank amino group of a specific lysine residue at delirium the active of the enzyme  symptoms can be relieved by niacin  Equilibrium of Transamination Reactions administration  equilibrium constant is near 1 which allows  Cystinuria the reaction to function in both ways  most common genetic error of amino acid  amino acid degradation (α-amino group transport removal)  autosomal recessive defect in kidney tubular  amino acid biosynthesis (addition of amino reabsorption of the basic amino acids resulting in groups to the carbon skeletons of α- high levels of their excretion ketoacids  relatively insoluble cystine in the urine  Diagnostic Value of Plasma Aminotransferases  normally intracellular enzymes causes cell  most of the symptoms are due to stone formation damage and release of the enzymes to the  quantitative urinary amino acid analysis confirms plasma the diagnosis  Liver Disease is characterized by increased  Treatments to reduce cystine crystal deposition plasma levels (ALT > AST) in nearly all include Urine Dilution and Penicillamine liver diseases; hepatocellular damage results  Glycinuria (Iminoglycinuria) to elevated serum bilirubin  transport system defect for Imino Acids (Proline, o ALT is more specific for liver Hydroxyproline) & Glycine disease  decreased renal tubular absorption o Liver contains greater amount of  benign disorder with no clinical abnormalities AST so it is more sensitive for liver disease o Seen in severe viral hepatitis, toxic NITROGEN REMOVAL from AMINO ACIDS injury, and prolonged circulatory collapse A. α-Amino Groups  Nonhepatic Disease is characterized by  keep amino acids locked away from oxidative breakdown increased plasma levels (ALT < AST) o Seen in Myocardial infarction,  Transamination (Funnelling of Amino Groups to Muscle disorders Glutamate)  1st step of amino acid catabolism  Oxidative Deamination  transfer of an amino group from an amino acid to  Results to liberation of amino group as free ammonia an α-keto acid  new amino acid + new α-keto  Site is mitochondrial matrix acid  Occurrence is primarily in liver and kidney Trans Finals 7a | ABACCO, ALDERITE, ASISTIN, BALANZA, BAYAS, BIANG 4 of 5  1A BIOCHEMISTRY AMINO ACIDS: NITROGEN DISPOSAL (Part 1) DR. B. JANDOC   Provide Ketoacids and Ammonia  Control reversible reaction driven by need for TCA intermediates  low energy (GDP, ADP) activates  high energy (GTP, ATP) inhibits  Glutamate Dehydrogenase  Regenerate Ketoglutarate from glutamate  Glutamate is the only amino acid that undergoes rapid oxidative deamination catalyzed by glutamate dehydrogenase  Coenzymes include o Oxidized NAD (NAD+) which is used primarily in oxidative deamination (simultaneous loss of ammonia coupled with oxidation of the carbon skeleton) o Oxidized NADP (NADP+) which is used in reductive amination (simultaneous gain of ammonia coupled with reduction of the carbon skeleton) o NADPH-NADP+ Ratio in Normal Liver Conditions: -reduced NADP (NADPH) to NADP+ (high) -reduced NAD (NADH) to NAD+ (low)  Direction of Reactions depends on o Relative Concentrations of glutamate, ketoglutarate, ammonia o Ratio of Oxidized to Reduced Coenzymes  Allosteric Regulators o Inhibitors include ATP, GTP, and NADH o Activators include ADP & GDP  Alternative Mechanisms for Amino Acid Deamination  Amino Acid Oxidases  L- and D-amino acid oxidases occur in the kidneys and liver  D-Amino Acid Oxidase o FAD-dependent enzyme o catalyzes oxidative deamination of D-amino acids resulting to formation of ketoacids  Direct Deamination by Dehydratases o Cofactor is pyridoxal phosphate o Serine Dehydratase, Threonine Dehydratase  Ammonia Transport to the Liver  2 Mechanisms  First Mechanism is found in most tissues o Glutamine Synthetase o Glutaminase  Second Mechanism o used primarily by muscles o involves transamination of pyruvate to alanine Trans Finals 7a | ABACCO, ALDERITE, ASISTIN, BALANZA, BAYAS, BIANG 5 of 5