Protein Metabolism

A. Sources and Uses of Amino Acids
B. Nutritional (Dietary) Requirements for Amino Acids

C. Digestion, Adsorption and Excretion
D. Nitrogen Balance

E. Endogenous Amino acid Degradation and Urea Cycle
F. Hepatic Glutamine Metabolism G.Biosynthesis and Utilization of Various Amino Acids in Specialized Biosynthetic Pathways

A. Sources and Uses of Amino Acids

Sources : Proteins in the diet provide both essential and nonessential amino acids
Uses : 1.Protein synthesis 2.Nitrogen and carbon source of general and special product biosynthesis 3.Energy source a.glucogenic(those that can be used for the synthesis of glucose) b.ketogenic(those whose metabolism leads to ketone bodies)

Amino acid roles:
1) protein monomeric units (primary purpose) 2) energy metabolites (about 10% of energy) 3) precursors of many biologically important nitrogen containing compounds such as: a) HEME b) physiologically active AMINES [(nor)epinephrine, dopamine, GABA (g-aminobutyric acid), serotonin, histamine] c) GLUTATHIONE e) NUCLEOTIDES f) nucleotide COENZYMES

an extremely stable compound: the N ΞN triple bond has a bond energy of 945kJ/mol. All organisms need bioavailable sources of nitrogen for proteins and nucleic acids. . The major form of nitrogen in the atmosphere is N2.Amino acid metabolism is intimately intertwined with nitrogen acquisition.

called diazatrophs. where they convert N2 to NH3 (ammonia) in a process called NITROGEN FIXATION: NITROGENASE N2 + 8 H+ + 8 e. Diazatrophs of the genus Rhizobium live symbiotically in the root nodules of legumes.N2 is converted to metabolically useful forms (is "fixed") only by a few species of prokaryotes.+ 16 ATP + 16 H2O → 2 NH3 + H2 + 16 ADP + 16 Pi .

NO3 . less than 1% of N entering the biosphere comes from N fixation.+ 8 H+ + 6 e.* But.. and certain bacteria in a two-step pathway: (1) The 2-electron reduction of nitrate to nitrite: NO3.→ NH4+ + 2 H2O (2) . Another oxidized form of nitrogen.. various fungi.+ 2 H+ + 2 e.to NH4 + (ammonium ion) occurs in green plants. NITRATE ASSIMILATION: * The reduction of NO3.→ NO2. It is converted to NH4 + through.(nitrate ion) is also found in the soils and oceans.+ H2O This is followed by the 6-electron reduction of nitrite to ammonium: NO2.

. such as amino acids and proteins. to provide this essential nutrient.No animals are capable of either N-fixation or nitrate assimilation. so they (we!) are totally dependent on plants and microorganisms for the synthesis of organic nitrogenous compounds.

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They must be hydrolyzed by a group of proteases and peptidases to amino acids.Digestion.C. Dietary proteins can not be absorbed directly from the intestine. The first major site of digestion is in the stomach by the action of pepsin. Adsorption and Excretion Digestion.trypsin and chymotrypsin) and intestinal peptidases functioning in the intestine then complete the hydrolytic process.g. dipeptides and tripeptides. then by the action of pancreatic enzymes(e. .

The amino acids and small peptides are transported into the intestinal cells by a family of amino acid specific transports many of which require Na+. . Absorption.

stomach pepsin pancreas to intestinal small intestine wall Trypsin Chymotrypsin carboxypeptidase A carboxypeptidase B dipeptidases elastase .

Nitrogen is also constantly being lost from the body in a variety of different forms. . The principal excretory nitrogen product in mammals is urea (about 12-20 g urea nitrogen/day).Excretion.

 Other excretory products include ammonium ion. uric acid and creatinine. The amounts of each of these is dependent of the metabolic state of the individual: Ureotelic-excrete urea (mammals) Uricotelic-excrete uric acid (birds.      . insects) Ammonotelic-excrete ammonium ion (fish) Some animals switch from one form to another during development (more about that later).

Nitrogen balance  Protein content of adult body remains remarkably constant  Protein constitutes 10-15% of diet  Equivalent amount of amino acids must be lost each day .

OVERVIEW OF AMINO ACID METABOLISM ENVIRONMENT ORGANISM Ingested protein 1 Biosynthesis 2 3 Protein a AMINO ACIDS c Degradatio n (required) (ketogenic) Urea acetoacetate acetyl CoA b c Purines Pyrimidines Porphyrins Nitrogen Carbon skeletons Used for energy (glucogenic) pyruvate α-ketoglutarate succinyl-CoA fumarate oxaloacetate .

The mechanisms of utilization of those consumed in excess for the purpose of synthesizing the deficient ones is part of the dynamic metabolism of nitrogen metabolism. therefore they take in some amino acids in excess of their needs. .Mammals cannot select the specific amino acids they have in their diet. Of course the essential amino acids cannot be made de novo in the animal cell.

 Amino acids. The carbon skeletons are oxidatively degraded for the production of energy or stored as carbohyrdrate. . are not excreted in any large amounts but are deaminated in one of several ways. which are not utilized for protein synthesis or other pathways of nitrogen utilization.   Ammonia produced is re-utilized or new amino acid synthesis or converted to urea for excretion.

Glutamine synthetase c.Oxidative (NAD an FAD dependent) b.Glutamate dehydrogenate b.Urea Cycle .Transamination(aminotransferases.Deamination a.E.Carbamoyl phosphate synthetaseI 4.Ammonia Assimilation a.Endogenous Amino Acid Degradation and Urea Cycle 1. transaminases) 2. Non-oxidative 3.

valine) occurs primarily in skeletal muscle  amino groups transferred to alanine and taken to liver for disposal via glucosealanine cycle Gluconeogenesis (in liver) Oxidised in Krebs Cycle From: Summerlin LR (1981) Chemistry for the Life Sciences.Fate of amino acids  If not required for protein synthesis amino groups removed   For most amino acids occurs primarily in liver For (leucine.  Carbon skeletons used for:    Amino groups used for   Synthesis of nonprotein nitrogen compounds disposed of via Urea Cycle . isoleucine. New York: Random House p 563.

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but 3 common reactions:  Transamination Deamination Formation of urea   .Amino acid metabolism  Metabolism of amino acids differs.

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What is produced if oxaloacetate is the acceptor? . producing GLUTAMATE and the new a-keto acid.Typical first transamination reaction: The usual AA acceptor is α-ketoglutarate.

. so produces…. X oxaloacetate X ASPARTATE .What is produced if oxaloacetate is the acceptor? It is one C shorter than a-ketoglutarate.

The second typical step in AA deamination involves transfer of the amino group from GLU to oxaloacetate. yeilding a-ketoglutarate and ASP: glutamate-aspartate aminotransferase .

TRANSAMINATION .

Biosynthesis of Nonessential Amino Acids  Transamination reactions   Allow extensive interconversion between nonessential amino acids Requires vitamin B6 as a coenzyme .

Transamination   Transfer of amino group from an amino acid to an α-keto acid Used to synthesize amino acids as needed  Some essential amino acids  Not lysine or threonine  Must have appropriate α-keto acid in diet Pyridoxal phosphate (PLP)  Requires vitamin B6 in coenzyme form   Catalyzed by amino transferases .

Amino Acid Transamination α-Keto Acid PLP amino Amino transferase amino α-Keto Acid α-Keto Acid α-Keto Acid α-Amino Acid .

Transamination .

Amino Acid Interrelationships  Methionine can be converted to Cys  If too little Cys in diet. Met is converted to Cys and Met becomes deficient  Up to 50% of Cys „requirement‟ met through Met  Phe can be converted to Tyr  Requirement is typically stated for Phe + Tyr + NH3 O O– NH3+ HO C C H2 H C O O– C C H2 H C .

Protein Catabolism  Occurs when  Dietary protein exceeds protein requirements of body   Normal situation in true carnivores Abnormal in omnivores and herbivores   Composition of absorbed amino acids is unbalanced Gluconeogenesis is increased .

excrete the nitrogen as urea .Protein Catabolism  Some net catabolism of body proteins occurs at all times  Expressed as urinary nitrogen excretion  Use the carbon backbone for energy.

Urinary Nitrogen Excretion LIVER Amino acids CO2 NH3 Urea Blood keto acids KIDNEY Urea Urine .

Deamination   Removal of amino group from an amino acid with no transfer Produces ammonia and α-keto acid   Ammonia removed by urea cycle α-keto acid is metabolized via several potential pathways  Pyridoxal phosphate (PLP) required (B6) .

Deamination amino Dehydratase PLP α-Keto Acid α-Keto Acid H2O + NH4 .

acetyl-CoA) Used as energy source in various tissues . not all amino acids In liver (and kidney)   Lipogenesis (fatty acid biosynthesis) Ketogenesis   Ketone bodies (acetoacetate.Use of Keto Acids for Energy  Keto acids can   Enter the TCA cycle and be broken down to CO2 and H2O with release of energy Be used for gluconeogenesis   Some.

Ketogenic Amino Acids  Leucine and isoleucine  Converted to acetoacetate or acetyl CoA in liver  Fuel for other tissues .

0 Cost ¢/Mcal 2.102 3.42 3.4 150.38 ME Mcal/ton 3.30 4.066 Cost $/ton 71.Use of Amino Acids for Energy  Not economical  Energy feeds are less expensive (per kcal) than protein feeds Item Corn SBM CP % 8 48 ME Mcal/kg 3.89 .

Disposal of NH3  NH3 is very toxic and must be detoxified and excreted from the body    Fish: Mammals: Birds: NH3 Urea Uric acid  Synthesis of uric acid  Same pathway as for purines Detoxifies NH3 to urea Synthesizes arginine  Synthesis of urea—the urea cycle   UREA .

Urea Cycle .

Urea Cycle  Overall reaction 2 NH3 + CO2 O ||   H2N–C–NH2 + H2O • Energy required (3 ATP) • Urea diffuses from liver cells to body fluids • Excreted by the kidneys .

LS (1988) Biochemistry (3rd Ed). p500   Urea excreted in urine .Urea cycle  Ammonia is toxic  Readily ionises to ammonium ion NH4+  NH4+ converted to urea in liver (urea cycle)  Urea contains 2 x NH2  One from NH4+ One from aspartate From: Stryer. New York: WH Freeman & Co.

UREA CYCLE mitochondri a cytosol Function: detoxification of ammonia (prevents hyperammonemia) .

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.O H 2N C NH2 urea Most terrestrial land animals convert excess nitrogen to urea. prior to excreting it. Urea is less toxic than ammonia.

L-citrulline 49 .) Severe neurological defects in neonates Treatment:    Stop protein intake Dialysis Increase ammonia excretion: Na benzoate. excreted by kidney Principal method for removing ammonia Hyperammonemia:    Defects in urea cycle enzymes (CPS.Urea Formation    Occurs primarily in liver. etc. L-arginine. OTC. Na phenylbutyrate.

/Dl Elevated in renal insufficiency Decreased in hepatic failure 50 .Blood Urea Nitrogen    Normal range: 7-18 mg.

severe mental retardation results.Hereditary deficiency of any of the Urea Cycle enzymes leads to hyperammonemia . If not treated immediately after birth. . especially to the brain. Elevated ammonia is toxic.elevated [ammonia] in blood. Total lack of any Urea Cycle enzyme is lethal.

1 in 15. noradrenaline. thyroxine and melanin synthesis .Conditionally Essential Amino Acids  Amino acids that can become essential in certain physiologic conditions    Example: taurine in cats Example: proline in young pigs Example: tyrosine becomes essential in people with “phenylketonuria (PKU)”   PKU.000 babies Hydroxylation of phenylalanine normally forms tyrosine  Tyrosine important in adrenaline.

Phenylketonuria (PKU)  Normal situation: Phenylalanine (essential aa) Tyrosine (nonessential aa)  In PKU:  Phenylalanine builds up Can cause mental retardation Condition inherited from parents (genetic) Phenylalanine (essential aa) Tyrosine (nonessential aa) .

hair and eyes than siblings           Delayed mental and social skills Head size significantly below normal Hyperactivity Jerking movements of the arms or legs Mental retardation (severe if not diagnosed and treated early) Seizures Skin rashes Tremors Unusual positioning of hands Phenylalanine important in synthesis of melanin .PKU Symptoms   VERY FEW symptoms if diagnosed early and diet strictly regulated IF NOT:  Lighter skin.

eggs No aspartame (NutraSweet) Fish oil supplements  Hard to get enough essential fatty acids on low phenylalanine diet  Iron supplements .PKU Treatment  EXTREMELY low phenylalanine intake   Diet for life Special low-phenylalanine infant formula  Used for life    Low or no milk.

(2000) .Boisen et al.