Protein Metabolism PDF

Protein and Amino Acid Metabolism Remedios P. Santos, M.D.  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 libera...

Protein and Amino Acid Metabolism Remedios P. Santos, M.D.  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.  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, 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.  Two major pathways that degrade intracellular proteins of eukaryotic cells 1) LYSOSOMAL DEGRADATION Lysosomes have a selective pathway, which is activated only after a prolonged fast, that imports and degrades cytosolic proteins containing the pentapeptide KFERQ (Lys-Phe-Glu-Arg-Gln) or a closely related sequence. 2) UBIQUITIN - Proteins are marked for degradation by covalently linking them to ubiquitin. Whether or not a given protein is derivatized by ubiquitin depends on which aminoacyl residue is present at its amino terminal. Reaction with ubiquitin is retarded by amino terminal methionyl or seryl residue and is accelerated by amino terminal aspartyl or arginyl residues. Overall Sources & Utilization of Amino Acids  Central Role of the Liver in Amino Acid Metabolism Protein Synthesis of other Delivery to other Synthesis N-ctg Compounds organs of a balanced mixture of amino acids Catabolism of both Synthesis of C-Chain and N of non-essential amino acids amino acids  Classification of amino acids from a nutritional point of view  Essential or indispensable amino acids - can not be synthesized by the body so they must be provided in the diet - Trp Met His Leu Val Arg Ile Lys Thr Phe Non-essential or dispensable amino acids - can be synthesized by the body so they need not be provided in the diet.  3 enzymes that occupy central positions in amino acid biosynthesis: - glutamate dehydrogenase - glutamine synthetase - aminotransferases The combined effect of these 3 enzymes is to transform ammonium ion into the alpha-amino nitrogen of various amino acids.  Amino Acid Catabolism A. Removal of the -NH2 group 1. Oxidative deamination 2. Transamination 3. Non-oxidative deamination B. Decarboxylation C. Oxygenation D. One-Carbon transfer  Oxidative Deamination  Overall Reaction Amino Acid Keto acid + NH3  Enzymes involved 1. Glutamate dehydrogenase – Glu is the only amino acid that undergoes oxid. deamination at an appreciable rate in mammalian tissues. COOH H2O COOH COOH (CH2)2 + NAD+ (CH2)2 (CH2)2 CH-COOH NH=C-COOH O=C-COOH NH2 -Iminoglutaric -Ketoglutarate acid + NH4 + NADH2 Glutamate  Oxidative Deamination (continued) 2. L-amino acid oxidase L-a.a. + H2O + E-FMN -Keto acid + NH3 + E-FMNH2 Ex. Aspartic acid Oxaloacetate + NH3 Alanine Pyruvate + NH3 3. D-amino acid oxidase D-a.a. + H2O + E-FAD -Keto acid + NH3 + E-FADH2 Zellweger Cerebrohepatorenal syndrome (CHRS) - Abnormal neural development, generalized aminoaciduria, absence of peroxisomes in a liver biopsy. The aminoaciduria is due to deficient amino acid oxidase activity.  Non-Oxidative Deamination  Enzymes involved 1. Amino acid dehydrases  Requires B6PO4 as cofactor  Hydroxy amino acids (serine, threonine, tyrosine, homoserine) -H20 OH-CH2-CH-COOH CH2=C-COOH CH3-C-COOH NH2 NH2 NH Imino acid Serine Intermediate ±H2O NH3 + CH3 -C-COOH O Pyruvic acid  Non-Oxidative Deamination (continued) 2. Amino acid desulfhydrases  Requires B6PO4 as cofactor  Sulfur-containing amino acids (cysteine, homocysteine, methionine) -H2S S-CH2-CH-COOH CH2=C-COOH CH3-C-COOH NH2 NH2 NH Cysteine Imino acid NH3 + CH3 -C-COOH O Pyruvic acid  Transamination  Enzyme: Transaminase or aminotransferase  General reaction  Example  Enzyme: Aspartate aminotransferase or Glutamate-oxaloacetate transaminase  Co-enzyme: Pyridoxal Phosphate (Vitamin B6 Carrier between the amino acid and the keto acid Amino acids that do not undergo transamination – Lysine, Threonine, Proline, OHProline  DECARBOXYLATION  Leads to the formation of Biogenic Amines.  Examples: - Serotonin or 5-hydroxytryptamine – Trp - Histamine – Histidine - Catecholamines – Dopamine, norepinephrine, epinephrine – Tyrosine and Phenylalanine  Ways of Detoxifying Ammonia 1. Reversal of the glutamate dehydrogenase rxn Glutamic acid -Ketoglutarate + NH3 2. Glutamine formation Glutamine Glutamic acid + NH3 Glutamine Synthetase 3. Urea formation 4. Asparagine formation Aspartic acid + NH3 Asparagine  Urea Cycle  Enzymes 1. Carbamoyl PO4 Synthetase I 2. Ornithine Transcarbamoylase 3. Argininosuccinate synthetase 4. Argininosuccinase 5. Arginase  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 1. Hyperammonemia Type I – Carbamoyl phosphate synthetase I 2. Hyperammonemia Type II – Ornithine transcarbamylase 3. Citrullinemia – Argininosuccinate synthetase 4. Argininosuccinate aciduria – Argininosuccinase 5. Arginemia - Arginase  HHH syndrome – Hyperornithinemia, hyperammonemia and homocitrullinuria - results from mutation of the ORNT1 gene that encodes the mitochondrial membrane ornithine transporter. Failure to import cytosolic ornithine into the mitochondrial matrix renders the urea cycle inoperable with consequent hyperammonemia and the accumulation of cytosolic ornithine results in hyperornithinemia. Therapy for urea cycle enzyme deficiencies has a 3-fold basis 1. To limit protein intake and potential build-up of ammonia - limit ingestion of amino acids - give levulose to promote excretion of ammonia 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  Metabolic Fates of the Keto Acids 1. Synthetic pathway -Ketoacid + NH3 -amino acid 2. Glucogenic pathway 3. Ketogenic pathway 4. Miscellaneous pathways  Purely Ketogenic amino acid – Leucine Metabolic Fates of the Keto Acids  Both glycogenic and  Glycogenic amino ketogenic acids  Ile  Ala Val  Phe  Arg Met  Tyr  Asp Gly  Trp  Asn His Lys  Cys Ser  Glu Thr  Gln Pro Catabolic Disposition (Fates) of Carbon Chains of Amino Acids  Metabolic Pathway of Phe/Tyr  Enzymes 1. Phenylalanine monooxygenase or phenylalanine oxidase or phenylalanine hydroxylase 2. Homogentisate 1,2-dioxygenase 3. Tyrosinase  Phenylketonuria 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  Two Types of Tyrosinemias 1. 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 R - Richner-Hanhart syndrome - deficiency of tyrosine aminotransferase leading to accumulation and excretion of Tyr and metabolites. S/S include eye and skin lesions and mental retardation.  Tryptophan 1. 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 ALA * main pathway deficiency) Hydroxyanthranilate Niacin Acetoacetyl CoA  Tryptophan 2. Conversion to serotonin Tryptophan Hydroxytryptophan decarboxylase CO2 * neurotransmitter Serotonin (5-hydroxytryptamine) * vasoconstrictor monoamine oxidase * stimulates smooth NH3 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 INBORN ERRORS OF TRP METABOLISM 1. 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. - symptoms are caused by secretion by the tumor of serotonin, prostaglandins, and other biologically active substances. 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) 3. Blue Diaper Syndrome - impaired intestinal and renal absorption of tryptophan - familial disorder characterized by hypercalcemia, nephrocalcinosis, and indicanuria. Dietary Trp Skatole and Indole (Large Intestines) absorbed then goes to liver Indican Excreted in urine  CATABOLISM OF HISTIDINE HISTIDINE Histidase Histidinemia NH4+ UROCANIC ACID 4-Imidazolone-5-propionate Urocanase H2O Imidazolone propionate hydrolase Figlu Excretion N-Formiminoglutamate (FIGLU) test – test for THFA Glutamate formimino Folic acid transferase deficiency N5-Formimino THFA GLUTAMIC ACID pyruvate Transaminase Alanine -Ketoglutarate  -KG Transamination Glu -Ketoisovaleric acid CoA, NAD+ -Ketoisovaleric acid dehydrogenase NADH, CO2 Isobutyryl CoA Acyl CoA DH Methylacrylyl CoA Methylmalonic acid semialdehyde Propionyl CoA Succinyl CoA  Leucine -KG Transaminase Glu -Ketoisocaproic acid CoA, NAD+ -Ketoisocaproic dehydrogenase NADH, CO2 Isovaleryl CoA Acyl CoA DH ß-Methylcrotonyl CoA ß-Hydroxy-ß-methyl Glutaryl CoA (HMG-CoA) Acetyl CoA Acetoacetic acid  -KG Transamination Glu -Keto-ß-methylvaleric acid CoA, NAD+ -Keto-β-methylvaleric dehydrogenase NADH, CO2 -Methylbutyryl CoA Acyl CoA DH Tiglyl CoA -Methylacetoacetyl CoA Propionyl CoA Methylmalonyl CoA Succinyl CoA Metabolic Disorders of Branched-chain amino acid catabolism 1. Maple Syrup Urine disease – MSUD - also called branched-chain ketonuria - absence or deficiency of -keto acid decarboxylase - 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.  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 1. Conversion to -Ketobutyric acid by threonine dehydratase -Ketobutyric acid propionic acid glucose 2. Cleavage to glycine and acetaldehyde by threonine aldolase  Pathways for Threonine Degradation (continued) 3. Dehydrogenation and decarboxylation to yield aminoacetone Aminoacetone + O2 2-Ketopropanol Pyruvate Aldehyde DH Acetaldehyde Acetyl CoA NAD+ CoA NADH  Methionine  Inborn Errors 1. Homocystinuria  Deficiency of cystathionine synthetase 2. Cystathioninuria  Deficiency of cystathionase  Two Principal Catabolic Pathways of Cysteine 1. Direct oxidative pathway-  Cysteine sulfinate pathway Cysteine Cysteine sulfinate pyruvate 2. Transamination pathway-  3-Mercaptopyruvate pathway Cysteine 3-Mercaptopyruvate pyruvate  Inborn Errors 1. Cystinuria (Cystine-Lysinuria)  Considered to be due to a renal transport defect affecting renal reabsorptive mechanisms for 4 amino acids – cystine, lysine, arginine and ornithine  Manifested by increased urinary excretion of cystine, lysine, arginine and ornithine  Cystine is insoluble  may precipitate in kidney tubules and form cystine calculi  Inborn Errors (continued) 2. Cystinosis (Cystine Storage Disease)  Primary defect: Impaired lysosomal function  Signs and symptoms  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 1. Major Route – Glycine synthase Glycine + FH4 + NAD N5N10 –Methylene FH4 + CO2 + NH3 + NADH + H+ 2. Conversion to serine by serine hydroxymethyl transferase 3. Oxidative deamination by glycine oxidase to yield glyoxylic acid H2O Glycine H2O2 oxidase Glycine Glyoxylic acid O2 NH3  Metabolic Pathways for Glycine  Heme synthesis  Synthesis of purines  forms positions C4, C5, N7 of the purine ring  Constituent of glutathione  Conjugates with cholic acid to form glycocholic acid  Conjugates with benzoic acid to form hippuric acid  Synthesis of creatine 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 (con’t) transaminase  -Ketoglutarate Glutamic acid amino acid keto acid Histidine Figlu Glutamic acid Arginine Ornithine Glutamic acid Proline Pyrroline-5-carboxylic acid Glutamic acid  Synthesis of Glutamine Glutamate NH4+ Mg-ATP glutamine synthetase Mg-ADP + Pi Glutamine Glutamine synthetase reaction  Synthesis of Aspartic Acid Transamination Oxaloacetate Aspartic acid Glutamic acid -ketoglutarate  Asparagine Synthesis Aspartate Glutamine Mg-ATP asparagine synthetase Mg-AMP + PPi Glutamic acid Asparagine Asparagine synthetase reaction  Synthesis of Alanine Pyruvate Glutamate (or Asp) aminotransferase &-ketoglutarate (or oxaloacetate) Alanine Transamination 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 (con’t) 2) CO2 + NH3 + N5,N10-methylene FH4 Pyridoxal PO4 Glycine synthase Glycine + FH4 + NAD 3) Threonine Glycine + Acetaldehyde cleavage enzyme Synthesis of Glycine (con’t) Glyoxylate Glutamate or Alanine glycine aminotransferase &-KG or pyruvate Glycine Synthesis of Glycine (con’t) Choline Betaine aldehyde Betaine Dimethylglycine Sarcosine Glycine  Synthesis of Proline Glutamate NADH H20 Glutamate semialdehyde H20 Pyrrolidine-5-carboxylate NADH Proline Reversal of proline catabolism  Synthesis of Cysteine Serine + Homocysteine (fr. Methionine) H20 Cystathionine H20 Cysteine + Homoserine  Synthesis of Tyrosine Phenylalanine Tetrahydrobiopterin NADP phenylalanine hydroxylase Dihydrobiopterin NADPH O2 H20 Tyrosine Synthesis of Non-essential amino acids 1. Glycine Serine 2. Proline Glutamic acid 3. Arginine Glutamic acid 4. Histidine Glutamic acid 5. Phenylalanine Tyrosine 6. Threonine Glycine 7. Methionine Cysteine 8. Aspartic acid Asparagine 9. Glutamic acid Glutamine 10. Pyruvate Alanine 11. Oxaloacetate Aspartic acid 12. -ketoglutarate Glutamic acid 13. 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  2. 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  3. Creatine Arginine + Glycine Arg-gly transaminidase (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  4. Porphyrins Succinyl CoA + Glycine δ-aminolevulinate synthase δ-Aminolevulinic acid (ALA) Porphobilinogen Uroporphyrinogen III Protoporphyrin III (IX) Fe2+ Ferrochelatase Heme Hemoglobin Myoglobin Cytochrome  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 Carnosine – composed of ß-alanine and histidine; in humans it is found in skeletal muscle and brain, particularly in the primary olfactory pathways; acts as neurotransmitter Anserine – related to carnosine; composed of ß-alanine and methylated His; occurs in the skeletal muscle of birds and some mammals excluding humans.  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  Methionine con’t. 3. Creatine phosphate - high energy compound that can phosphorylate ADP to form ATP - provides energy for muscle contraction 4. Choline SAM Ethanolamine Choline - important component of acetylcholine (neurotransmitter), lecithin & sphingomyelin (membrane phospholipids)  Methionine con’t. 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, e.g., fatty acyl CoA 2. Taurine - conjugates with bile acids to form bile salts e.g., 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  Arginine con’t. 2. Creatine - arginine is the formamidine donor for creatine synthesis 3. Putrescine, spermidine & spermine Arg→ Ornithine→ Putrescine→ Spermidine→ Spermine Putrescine- a polyamine found 1st in decaying animal tissues but now known to occur in almost all tissues and in cultures of some bacteria. Spermidine and spermine - polyamines 1st found in human semen but now known to occur in almost all tissues in association with nucleic acids.  Derivatives of Phe & Tyrosine 1. Triiodothyronine (T3) and Thyroxine (T4) 2. Melanin 3. Norepinephrine and epinephrine  Derivative of Glutamic Acid 1. Gamma-aminobutyric acid (GABA) Glutamic acid glutamate decarboxylase; PLP 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) 2. Indole and skatole 3. Melatonin 4. Niacin Glucose – Alanine Cycle  Functions of Glucose – Alanine Cycle 1. To carry amino groups from skeletal muscle to the liver to be converted to urea. 2. To provide the working muscle with blood glucose made by the liver from the carbon backbone of alanine. 3. 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