Amino Acid Metabolism and Urea Cycle PDF

Amino Acid Metabolism Urea Cycle The chemical transformations of amino acids are distinct from those of carbohydrates or lipids in that they involve the element NITROGEN. Excess dietary amino acids are not simply excreted but are converted to common metabolites that are precursors of glucose, fatt...

Amino Acid Metabolism Urea Cycle The chemical transformations of amino acids are distinct from those of carbohydrates or lipids in that they involve the element NITROGEN. Excess dietary amino acids are not simply excreted but are converted to common metabolites that are precursors of glucose, fatty acids, and ketone bodies and are therefore metabolic fuels.  PROTEIN TURNOVER It is the continuous degradation and resynthesis of all cellular proteins. Each day, humans turn over 1 – 2 % of their total body protein, principally muscle protein. Of the liberated amino acids, 75 – 80 % are reutilized for new protein synthesis. The nitrogen of the remaining 20 – 25 % forms urea. The carbon skeletons are then degraded to amphibolic intermediates.  INTRACELLULAR PROTEIN DEGRADATION The components of living cells are constantly turning over. Proteins have life times that range from as short as a few minutes to weeks or more. In any case, cells continuously synthesize proteins from and degrade them to amino acids. This seemingly wasteful process has 3 functions, namely: to store nutrients in the form of proteins and to break them down in times of metabolic need, processes that are most significant in muscle tissue. To eliminate abnormal proteins whose accumulation would be harmful to the cell. To permit the regulation of cellular metabolism by eliminating superfluous enzymes and regulatory proteins. The susceptibility of a protein to degradation is expressed as its HALF-LIFE. - The time required to reduce its concentration to 50% of its initial value - Half-lives for liver proteins range from under 30 minutes to over 150 hours. - Proteins with short half-lives usually have PEST sequences – regions rich in the amino acids Proline (P), Glutamate (E), Serine (S), and Threonine (T), which target them for rapid degradation. Proteins with half-lives over 100 hours include aldolase, lactate dehydrogenase, and cytochromes. The most rapidly degraded enzymes all occupy important metabolic control points (rate-limiting enzymes), whereas the relatively stable enzymes have nearly constant catalytic activities under all physiological conditions. The rate of protein degradation in a cell varies with its nutritional and hormonal state. Ex. Under conditions of nutritional deprivation, cells increase their rate of protein degradation so as to provide the necessary nutrients for indispensable metabolic processes.  Protein degradation 2 major enzyme systems inside the cell: ATP-dependent Ubiquitin-proteasome of cytosol ATP-independent degradation of the lysosomes Uses acid hydrolases  Protein degradation 2 major enzyme systems inside the cell: ATP-dependent Ubiquitin-proteasome of cytosol ATP-independent degradation of the lysosomes Uses acid hydrolases Overall Sources & Utilization of Amino Acids PROTEIN DIGESTION Mouth - no enzyme acting on proteins Stomach - Pepsin Small intestines: - Pancreatic enzymes: Trypsin, Chymotrypsin, Carboxypeptidase, Elastase - Enzymes produced by intestinal epithelial cells: aminopeptidases, other peptidases AMINO ACID ABSORPTION Amino acids are absorbed from the intestinal lumen in 3 ways: 1) thru secondary active Na+-dependent transport 2) thru facilitated diffusion 3) thru transport linked to the gamma- glutamyl cycle Central Role of the Liver in Amino Acid Metabolism Protein Synthesis of other N- Delivery to other Synthesis CTG Compounds organs of a balanced mixture of amino acids LIVER Catabolism of both Synthesis of C-Chain and N of non-essential amino acids amino acids  Metabolic Fates of the Keto Acids Synthetic pathway -Ketoacid + NH3 -amino acid Glucogenic pathway Ketogenic pathway Miscellaneous pathways Metabolic Fates [cont’d…] ▪ Ketogenic amino acid - Leucine Both glycogenic and ketogenic Trp Phe Tyr Lys Ile Glycogenic amino acids Ala Asn Gln Met Ser Arg Cys Gly Thr Val Asp Glu HisPro Metabolic Pathway of Phe/Tyr Enzymes: 1. Phenylalanine monooxygenase or phenylalanine oxidase or phenylalanine hydroxylase 2. Homogentisate 1,2- dioxygenase 3. Tyrosinase Phenylketonuria (PKU) Phe hydroxylase Phe + O2 Tyr + H2O THB DHB NADP+ NADPH + H+ THB – Tetrahydrobiopterin; electron donor DHB – Dihydrobiopterin NADPH – ultimate electron donor DHB reductase – enzyme that converts DHB to THB  Tyrosinemias Type 1 or Hepatorenal tyrosinemia - deficiency of fumarylacetoacetate hydrolase - accumulation of fumarylaceto-acetate and maleylacetate, both of which are alkylating agents, can lead to DNA alkylation and tumorigenesis. - more serious type; leads to liver failure, renal tubular dysfunction, rickets and polyneuropathy. 2. Type II or Oculocutaneous tyrosinemia - deficiency of tyrosine aminotransferase leading to accumulation and excretion of Tyr and metabolites. S/S include eye and skin lesions and mental retardation. - also known as Richner-Hanhart Syndrome Catabolic Disposition (Fates) of Carbon Chains of Amino Acids Metabolic Pathways of Tryptophan Degradation via kynurenine-anthranilate pathway Tryptophan tryptophan oxygenase or Trp pyrrolase N-formylkynurenine Kynurenine Vit B2 alternative pathway Hydroxykynurenine Xanthurenate kynureninase; Vit. B6 (elevated in B6 def. ) ALA * main pathway Hydroxyanthranilate Niacin Acetoacetyl CoA  Tryptophan … 2. Conversion to serotonin Tryptophan Hydroxytryptophan decarboxylase CO2 * neurotransmitter Serotonin (5-hydroxytryptamine) * vasoconstrictor * stimulates smooth NH3 monoamine oxidase muscle contraction 5-hydroxyindoleacetate * Elevated in carcinoid syndrome (Argentaffinoma)  Tryptophan … 3. Formation of melatonin in the pineal body Serotonin acetylase N-acetylserotonin methylase; SAM Melatonin MELATONIN - a sleep-inducing molecule - acetyltransferase [pineal gland and retina] - involved in the regulation of circadian rhythm, being synthesized mostly at night. Tryptophan induces sleep - ingestion of foods rich in Trp leads to sleepiness because serotonin is also sleep-inducing aside from melatonin. - reducing availability of Trp in the brain can interfere with sleep. - Trp availability is reduced when other amino acids compete with it for transport thru the BBB. -  concentration of other AA, after a high protein meal,  transport of Trp and induce wakefulness.  Inborn Errors of Trp Metabolism Hartnup disease - defect in the intestinal and renal transport of Trp - deficiency of Trp pyrrolase - S/S include pellagra-like skin rash, cerebellar ataxia, intellectual deterioration. 2. Carcinoid syndrome - associated with carcinoid tumors occurring in the small intestines, appendix, colon, stomach. - S/S are caused by secretion by the tumor of serotonin, prostaglandins, … - Serotonin is the most common secretory product of carcinoid tumors and measurement of urinary 5-HIAA levels is the most useful diagnostic test.  Inborn Errors of Trp Metabolism [cont’d] 3. Blue Diaper Syndrome - impaired intestinal and renal absorption of Trp - familial disorder characterized by hypercalcemia, nephrocalcinosis, and indicanuria. Dietary Trp Skatole and Indole (Large Intestines) absorbed then goes to liver Indican Excreted in urine  HISTIDINE Histidinemia Histidase NH4+ UROCANIC ACID 4-Imidazolone-5-propionate Urocanase H2O Imidazolone propionate hydrolase N-Formiminoglutamate (FIGLU) FIGLU Excretion test - test for Folic THFA Glutamate formimino acid deficiency transferase N5-Formimino THFA GLUTAMIC ACID pyruvate Catabolism of Transaminase alanine Histidine -Ketoglutarate  Valine -KG Transamination Glu -Ketoisovaleric acid CoA, NAD+ -Ketoisovaleric acid dehyrogenase NADH, CO2 Isobutyryl CoA Acyl CoA DH Methylacrylyl CoA Methylmalonic acid semialdehyde Propionyl CoA Succinyl CoA  Leucine Leucine -KG Transaminase Glu -Ketoisocaproic acid CoA, NAD+ -Ketoisocaproic dehyrogenase NADH, CO2 Isovaleryl CoA Acyl CoA DH ß-Methylcrotonyl CoA ß-Hydroxy-ß-methyl Glutaryl CoA (HMG-CoA) Acetyl CoA Acetoacetic acid  Isoleucine -KG Transamination Glu -Keto-ß-methylvaleric acid CoA, NAD+ -Ketoisovaleric acid dehyrogenase NADH, CO2 -Methylbutyryl CoA Acyl CoA DH Tiglyl CoA -Methylacetoacetyl CoA Propionyl CoA Methylmalonyl CoA Succinyl CoA  Metabolic Disorders of Branched-chain AA Maple Syrup Urine disease - also called branched-chain ketonuria - absence or deficiency of -keto acid dehydrogenase - S/S: odor of urine resembles maple syrup or burnt sugar; infant is difficult to feed, lethargic and may vomit; extensive brain damage may occur 2. Isovaleric Acidemia - deficiency of isovaleryl CoA dehydrogenase. - manifested by cheesy odor of the breath and body fluids, vomiting, acidosis and coma precipitated by excessive ingestion of protein.  Lysine L-amino acid Saccharopine oxidase DH L-Ketoaminocaproic Saccharopine acid Saccharopine H2O, NAD+ Pipecolic acid DH NADH, L-Glutamate L--Aminoadipic acid semialdehyde -Aminoadipic acid -Ketoadipic acid Glutaryl CoA Crotonyl CoA Acetoacetyl CoA Pathways for Threonine Degradation Conversion to -Ketobutyric acid by threonine dehydratase -Ketobutyric acid propionic acid glucose Cleavage to glycine and acetaldehyde by threonine aldolase Aldehyde DH Acetaldehyde Acetyl CoA NAD+ CoA NADH 3. Dehydrogenation & decarboxylation to yield aminoacetone Aminoacetone + O2 2-Ketopropanol Pyruvate Methionine Inborn Errors Homocystinuria - Deficiency of cystathionine synthetase Cystathioninuria - Deficiency of cystathionase  2 Principal Catabolic Pathways of Cysteine Direct oxidative pathway: Cysteine sulfinate pathway Cysteine Cysteine sulfinate pyruvate 2. Transamination pathway: 3-Mercaptopyruvate pathway Cysteine 3-Mercaptopyruvate pyruvate Inborn Errors of Cys Metabolism Cystinuria (Cystine-Lysinuria) - due to a renal transport defect affecting renal reabsorptive mechanisms for 4 AA: Cys, Lys, Arg and ornithine - S/S:  urinary excretion of Cystine, Lys, Arg, ornithine - Cystine is insoluble → precipitate in kidney → Cystine calculi 2. Cystinosis (Cystine Storage Disease) - Primary defect: Impaired lysosomal function - S/S: Deposition of cystine crystals in many tissues and organs particularly the reticuloendothelial system; impaired renal function leading to acute renal failure Pathways of Degradation of Glycine Major Route – Glycine synthase Glycine + FH4 + NAD N5N10 –Methylene FH4 + CO2 + NH3 + NADH + H+ Conversion to serine by serine hydroxymethyl transferase Oxidative deamination by glycine oxidase to yield glyoxylic acid H2 O Glycine H2O2 oxidase Glycine Glyoxylic acid O2 NH3 Glycine Cleavage system or Glycine synthase a multienzyme complex that resembles pyruvate dehydrogenase contains 4 protein components: 1. a PLP-dependent glycine decarboxylase (P Protein) 2. a lipoamide-ctg aminomethyl transferase (H protein) which carries the aminomethyl grp remaining after Gly decarboxylation 3. an N5,N10-methylene THF synthesizing enzyme (T protein), which accepts a methylene grp from the aminomethyl transferase. 4. a NAD+-dependent, FAD-requiring lipoamide dehydrogenase (L protein) Metabolic Pathways for Glycine Heme synthesis Synthesis of purines → forms positions 4, 5, 7 of the purine ring Constituent of glutathione [Gly, Cys, and Glu] Conjugates with cholic acid to form glycocholic acid Conjugates with benzoic acid to form hippuric acid Synthesis of creatine [Gly, Arg and Met] Biosynthesis of Nutritionally Nonessential Amino acids Nutritionally Nonessential Amino Acids Ala Asn Asp Cys Glu Gln Gly Pro Ser Tyr OHpro OHlys Synthesis of Glutamate Alpha-ketoglutarate NH4+ NADPH + H+ glutamate DH NADP+ H20 Glutamate Glutamate dehydrogenase reaction (reversal) Synthesis of Glutamate [cont…] transaminase -Ketoglutarate Glutamic acid amino acid keto acid Histidine Figlu Glutamic acid Arginine Ornithine Glutamic acid Proline Pyrroline-5-carboxylic acid Glutamic a. Synthesis of Aspartic acid Transamination Oxaloacetate Aspartic acid Glutamic acid -ketoglutarate Synthesis of Alanine Pyruvate Glutamate (or Asp) aminotransferase &-ketoglutarate (or oxaloacetate) Alanine Transamination reaction Synthesis of Glutamine Glutamate NH4+ Mg-ATP glutamine synthetase Mg-ADP + Pi Glutamine Glutamine synthetase reaction Synthesis of Asparagine Aspartate Glutamine Mg-ATP asparagine synthetase Mg-AMP + PPi Glutamic acid Asparagine Asparagine synthetase reaction Synthesis of Serine 3-Phosphoglycerate (from glycolysis) oxidation Phosphohydroxypyruvate transamination Phospho-L-serine dephosphorylation Serine Synthesis of Glycine & Serine Serine FH4 serine hydroxymethyl transferase Methylene FH4 Glycine Synthesis of Glycine [cont’d…] 2) CO2 + NH3 + N5,N10-methylene FH4 Pyridoxal PO4 Glycine synthase Glycine + FH4 + NAD 3) Threonine Glycine + Acetaldehyde cleavage enzyme Synthesis of Glycine [cont’d…] Glyoxylate Glutamate or Alanine glycine aminotransferase &-KG or pyruvate Glycine Synthesis of Glycine [cont’d…] Choline Betaine aldehyde Betaine Dimethylglycine Sarcosine Glycine Synthesis of Proline Glutamate NADH H2 0 Glutamate semialdehyde H 20 Pyrrolidine-5-carboxylate NADH Proline Reversal of proline catabolism Synthesis of Cysteine Serine + Homocysteine (fr. Methionine) H 20 Cystathionine H 20 Cysteine + Homoserine  Synthesis of Tyrosine Phenylalanine Tetrahydrobiopterin NADP phenylalanine hydroxylase Dihydrobiopterin NADPH O2 H20 Tyrosine Synthesis of Hydroxyproline and Hydroxylysine Proline Lysine &-ketoglutarate Pro Lys hydroxylase hydroxylase O2, Fe2+ vit. C O-succinate Hydroxyproline Hydroxylysine Synthesis of Non-essential amino acids Glycine Serine Proline Glutamic acid Arginine Glutamic acid Histidine Glutamic acid Tryptophan Alanine Phenylalanine Tyrosine Threonine Glycine Methionine Cysteine Aspartic acid Asparagine Glutamic acid Glutamine Pyruvate Alanine Oxaloacetate Aspartic acid -ketoglutarate Glutamic acid 3-phosphoglycerate Serine Specialized Products Derived From Amino acids  Derivatives of Glycine 1. Bile salts Cholic acid Chenodeoxycholic acid Glycine Glycocholate Glycochenodeoxycholate - Conjugation takes place in the liver - Bile salts are important in fat digestion and absorption - Also important in absorption of fat-soluble vitamins Derivatives of Glycine… Hippuric acid Glycine + Benzoic acid ATP CoASH AMP + PPi Benzoyl-CoA Glycine CoASH Hippuric acid - A detoxification reaction; can be used to test liver function  Derivatives of Glycine… 3. Creatine Arginine + Glycine Arg-gly transamidinase (kidney) Ornithine + Glycocyamine (guanidoacetate) SAM ATP Guanidoacetate methyltransferase SAH ADP (liver) Creatine PO4 Creatine Creatinine - H2o (muscle) - Creatine phosphate is a source of energy for muscle contraction Derivatives of Glycine… 4. Porphyrins Succinyl CoA + Glycine δ-aminolevulinate synthase δ-Aminolevulinic acid (ALA) Porphobilinogen Uroporphyrinogen III Protoporphyrin III (IX) Fe2+ Ferrochelatase Heme Hemoglobin Myoglobin Cytochrome Derivatives of Glycine… 5. Purines α-D-Ribose 5-phosphate Phosphoribosyl pyrophosphate (PRPP) 5-Phosphoribosylamine Glycine (C4, C5, N7) Glycinamide ribosyl-5-phosphate Inosine monophosphate (IMP) Adenosine monoPO4 Guanosine monoPO4 (AMP) (GMP)  Derivatives of Alanine 1. Coenzyme A - activates substances, e.g., fatty acyl CoA, succinyl CoA, propionyl CoA 2. Carnosine & Anserine (β-Alanyl Dipeptides) - activates myosin ATPase, chelate copper, enhance copper uptake Derivatives of Serine, Threonine and Tyrosine Phosphorylated derivatives (phosphoserine, phosphothreonine, phosphotyrosine) - regulate the activity of certain enzymes of lipid and carbohydrate metabolism - regulate properties of proteins that participate in signal transduction cascade, e.g., tyrosine kinase activity of insulin receptors Derivatives of Methionine 1. Polyamines (spermidine & spermine) - growth factors involved in cell proliferation - stabilize intact cells, subcellular organelles & membranes - have hypothermic and hypotensive properties - bear positive charges that enables them to associate with DNA and RNA 2. Epinephrine SAM Norepinephrine Epinephrine - catecholamine produced in the adrenal medulla - hormone with metabolic and cardiovascular effects Derivatives of Met [cont’d] 3. Creatine phosphate - high energy compound that can phosphorylate ADP to form ATP - provides energy for muscle contraction 4. Choline SAM Ethanolamine Choline - impt. component of acetylcholine (neurotransmitter), lecithin & sphingomyelin (membrane phospholipids)  Derivatives of Met [cont’d] 5. Carnitine SAM Lysine Carnitine - carrier of long-chain fatty acids from the cytosol to the mitochondrial matrix - important for beta oxidation of fatty acids Derivatives of Cysteine 1. Coenzyme A - source of thioethanolamine portion of CoA - component of activated compounds [fatty acyl CoA] 2. Taurine - conjugates with bile acids to form bile salts [taurocholate, taurochenodeoxycholate] 3. Cystine - found in proteins having disulfide bonds - stabilizes secondary and tertiary structures of proteins Derivatives of Histidine 1. Histamine - results from decarboxylation of histidine - secreted by mast cells as a result of allergic reactions or trauma - chemical messenger that mediates inflammatory and allergic reactions - stimulates gastric acid secretions - neurotransmitter in some parts of the brain - vasodilator 2. Ergothioneine, carnosine, anserine  Derivatives of Arginine 1. Nitric Oxide Arginine O2 NADPH NO synthase NADP Citrulline + NO - intercellular signaling molecule - neurotransmitter - smooth muscle relaxant - vasodilator Derivatives of Arg [cont’d...] 2. Creatine - Arg is the formamidine donor for creatine synthesis 3. Putrescine, spermidine & spermine Arginine arginase Ornithine + Urea Putrescine Spermidine Spermine  Derivatives of Tyrosine 1. Triiodothyronine (T3) and Thyroxine (T4) - thyroid hormones - regulate basal metabolism 2. Melanin Tyrosine tyrosine hydroxylase; tetrahydrobiopterin 3,4-Dihydroxyphenylalanine (dopa) tyrosinase Melanin  Derivatives of Tyr [cont’d...] 3. Norepinephrine and epinephrine Tyrosine tyrosine hydroxylase Dopa dopa decarboxylase Dopamine dopamine hydroxylase Norepinephrine methyltransferase; SAM Epinephine  Derivatives of Glutamic Acid 1. Gamma-aminobutyric acid (GABA) Glutamic acid glutamate PLP decarboxylase Gamma-aminobutyric acid (GABA) - inhibitory neurotransmitter - “ natural tranquilizer” of the brain  Derivatives of Tryptophan 1. Serotonin - stimulates smooth muscle contraction; vasoconstrictor - involved in normal and abnormal behavior - regulation of sleep and temperature - increased production in carcinoid (argentaffinoma) Tryptophan tryptophan hydroxylase; tetrahydrobiopterin 5-Hydroxytryptophan decarboxylase Serotonin monoamine oxidase 5-Hydroxyindoleacetic acid  Derivatives of Trp [cont’d...] 2. Indole and skatole - due to the action of bacteria on trp in the colon - responsible for unpleasant odor of the stool 3. Melatonin - used to induce sleep 4. Niacin - deficiency causes Pellagra Functions of Glucose – Alanine Cycle To carry amino groups from skeletal muscle to the liver to be converted to urea. To provide the working muscle with blood glucose made by the liver from the carbon backbone of alanine. Smooths out fluctuations in the blood glucose level in the periods between meals.  Nitrogen balance Energy, heme, purines, etc. Dietary Amino acid Excreted as urea, protein pool NH4+ Tissue Protein  Nitrogen Balance Negative nitrogen balance Due to metabolic stress Due to lack of an essential amino acid Inadequate dietary protein Starvation Positive nitrogen balance Growth Pregnancy Convalescence  Ways of Detoxifying Ammonia Reversal of the glutamate dehydrogenase rxn Glutamic acid -Ketoglutarate + NH3 Glutamine formation Glutamine Glutamic acid + NH3 synthetase Glutamine 3. Asparagine formation Aspartic acid + NH3 Asparagine 4. Urea formation Urea Cycle Enzymes: Carbamoyl PO4 synthetase Ornithine transcarbamoylase Argininosuccinate synthetase Argininosuccinase Arginase “Kreb’s bicycle” Two Forms of CPS CPS I : - uses ammonia as its nitrogen donor; - used in the urea cycle; - mitochondrial in location; - absolutely dependent on N-AGA for activity CPS II : - uses glutamine as its nitrogen donor; - involved in pyrimidine synthesis; - cytosolic in location; - not affected by N-AGA  INBORN ERRORS OF UREA SYNTHESIS Hyperammonemia Type I [Carbamoyl phosphate synthetase] 2. Hyperammonemia Type II [Ornithine transcarbamylase] 3. Citrullinemia [Argininosuccinate synthetase] 4. Argininosuccinate aciduria [Argininosuccinase] 5. Arginemia [Arginase] Therapy for urea cycle enzyme deficiencies has a 3-fold basis To limit protein intake & potential build-up of NH3 - limit ingestion of amino acids - give levulose to promote excretion of NH3 in feces - give antibiotics to kill NH3-producing bacteria 2. To remove excess ammonia - give cpds that bind covalently to amino acids and produce N-ctg molecules that are excreted in the urine [Ex. Benzoate + Glycine = Hippuric acid; Phenylacetate + Glutamine = Phenylacetylgln 3. Replace any intermediates missing from the urea cycle Treatment of Ammonia Intoxication (due to causes other than inborn errors) Aims of Treatment - elimination or treatment of precipitating factors - lowering of blood NH3 levels by decreasing absorption of proteins Modes of Therapy - low protein diet - Lactulose - Antibiotics  References 1. Harper’s Illustrated Biochemistry 30th Edition 2. Lehninger Principles of Biochemistry 5th Edition 3. Devlin Principles of Biochemistry With Clinical Correlation 6th Edition 4. Lippincott’s Biochemistry 5th Edition Thank you!