Amino Acid Metabolism & Urea Cycle PDF

Amino acid metabolism & the urea cycle Dr Donna Sellers 5_3_19  Session Learning Objectives LO1: Review the basic structure of amino acids and their roles in the body LO2: Be aware of the classification essential/non-essential and glucogenic/ketogenic amino acids LO3: O...

Amino acid metabolism & the urea cycle Dr Donna Sellers 5_3_19  Session Learning Objectives LO1: Review the basic structure of amino acids and their roles in the body LO2: Be aware of the classification essential/non-essential and glucogenic/ketogenic amino acids LO3: Outline the biosynthetic pathways for amino acids LO4: Explain the metabolic relationship between amino acid metabolism to central metabolism (eg. TCA cycle) LO5: Describe how the body degrades amino acids Describe how nitrogen is removed to produce carbon skeletons and the pathways involved Appreciate the role of the B-group vitamin pyridoxine (B6) in amino acid metabolism LO6: Outline the role of the urea cycle in processing ammonia to urea Trace the flow of N atoms, and outline how the cycle overlaps with the TCA cycle Useful texts: Baynes and Dominiczak, Chapters 18 and 19  Amino acids & their roles  Small biomolecules with unique chemical properties  Consist of a carbon skeleton plus an amine group (-NH ) 2  Numerous roles, including:  Building blocks for proteins  Precursors for other biologically important molecules (eg. haeme, neurotransmitters, peptide hormones,) glutathione, nucleotides, coenzymes  Fuel molecules (during fasting) (ketogenic/glucogenic amino acids)  Sources of amino acids include the diet & those synthesised by the body (non-essential/essential amino acids)  Involved in different pathways, tissues & organelles, i.e. very complex!  We will look only at the ‘highlights’ of amino acid metabolism  Excess dietary intake of protein is not stored as protein  always broken down to amino acids and used for the body’s needs via the intermediates pyruvate, oxaloacetate or acetyl CoA  so excess amino acids can be converted to FAT LO1: Review the basic structure of amino acids and their roles in the body  Basic Structure of an Amino Acid  R group distinguishes amino acids and gives them different physical & chemical properties  At physiological pH: - Amino groups are positively charged (NH +) 3 Carboxyl groups are negatively charged (COO -)  Metabolically, consider amino acids as consisting of a carbon skeleton (an alpha keto acid) plus an amino group, as by cleaving the amino group the remaining carbon skeleton can be recycled  Careful handling of the amino groups is required as free ammonia is toxic (hyperammonaemia) and can result in brain swelling, coma and ultimately death if untreated LO1: Review the basic structure of amino acids and their roles in the body The 20 common amino acids (found in proteins) LO1: Review the basic structure of amino acids and their roles in the body  Classification of Amino Acids  Amino acids can be classified in different ways:  Physico-chemical characteristics of their side chain  Metabolic fate of their carbon backbone (gluconeogenic/ketogenic)  Dietary (essential/non-essential) Glucogenic Ketogenic Most amino acids are glucogenic which means Amino acids with carbon skeletons that cannot be during their breakdown the remaining carbon converted to glucose (because they are broken skeleton can be converted to oxaloacetate and down to either acetyl CoA or acetoacetyl CoA) are then to glucose, if needed, via gluconeogenesis. shunted towards fat synthesis or ketosis These include Phenylalanine, tyrosine & tryptophan (aromatic) Lysine, threonine Leucine and Isoleucine (2 branched chain amino acids) LO2: Be aware of the classification essential/non-essential and glucogenic/ketogenic amino acids  Classification of Amino Acids  Humans can only synthesise about half of the amino acids  Those that can’t be synthesized are termed ‘essential’, meaning they’re required in the diet.  Certain foods contain varying amounts of the various essential amino acids Essential Non-essential amino acids amino acids Arginine* Alanine Histidine Asparagine Isoleucine Aspartate Leucine Cysteine Lysine Glutamate Methionine Glutamine Phenylalanine Glycine *Classed as Threonine Proline essential as although it can be Tryptophan Serine synthesised, not in Valine Tyrosine sufficient amounts LO2: Be aware of the classification essential/non-essential and glucogenic/ketogenic amino acids  Amino Acid Biosynthesis  Humans require the essential amino acids from the diet  To synthesise the non-essential amino acids we need 2 basic components: A source of carbon An amine group donor (NH3+) (a ketoacid) + Glutamate/Glutamine Aspartate Carbamoyl-P  The carbon skeleton of amino acids can be derived from a variety of metabolic precursors, from pathways such as glycolysis, TCA Cycle, pentose phosphate pathway or breakdown of important biomolecules  Once formed, amino acids can be used to synthesize protein or other important biomolecules (neurotransmitters, hormones, haeme etc) = essential amino acids in humans LO3: Outline the biosynthetic pathways for amino acids  Metabolic Fate of Dietary & Intracellular Protein  All cells can ‘remodel’ amino acids but most amino acid metabolism occurs in liver  Remodelling involves removing the amino group & recycling the carbon skeleton  To metabolise amino acids, the amino group must first be removed (deamination)  Toxic ammonia is converted, in the liver, to the less toxic compound urea, which is excreted in the urine LO4: Explain the metabolic relationship between amino acid metabolism to central metabolism eg TCA cycle  Catabolic fate of amino acids: Amino Acid Carbons Feed into TCA Cycle, Gluconeogenesis or Lipid Metabolism  Following deamination, the carbon chains of the amino acids are fed into central pathways of metabolism LO4: Explain the metabolic relationship between amino acid metabolism to central metabolism eg TCA cycle Metabolic Link between Major Biomolecules Dietary & body protein 1 Dietary amino acids contribute to tissue protein 2 Excess amine converted to urea via Urea Cycle 1 3 Carbon skeletons feed into TCA Cycle or converted to 4 acetyl CoA which is a 2 5 precursor for lipids (& ketone bodies) 5 4 Carbon skeletons can be broken down to pyruvate which 3 can be a precursor for a variety of molecules or as gluconeogenic substrate LO4: Explain the metabolic relationship between amino acid metabolism to central metabolism eg TCA cycle  Catabolism of amino acids: Deamination of Amino Acids  Most amino acids undergo deamination (removal of amine) in the liver  This can occur by the action of a range of enzymatic reactions:  aminotransferases  glutamate dehydrogenase (oxidative deamination)  glutaminase  Some amino acids are deaminated in skeletal muscle  But since muscle cannot make urea, the amino groups must be transported (SAFELY) to the liver:  Aminotransferases catalyse the transfer of amine groups from amino acids to amine acceptors (ie alpha keto acids like pyruvate) producing the amino acid, alanine  Alanine is released into the bloodstream and transported to the liver where it is transaminated to yield pyruvate. The amino group makes it’s way to urea.  Pyruvate is a precursor for gluconeogenesis. The liver releases glucose into the blood.  The Glucose-Alanine Cycle thus involves the transport of excess nitrogen from muscle (via alanine) to the liver,  In the liver it is converted into glucose via gluconeogenesis which is then exported from liver back to muscles for energy etc. LO5: Describe how the body degrades amino acids Describe how nitrogen is removed to produce carbon skeletons and the pathways involved Glucose-Alanine Cycle Four Key Amino Acids in Nitrogen Metabolism  Removal of amino group from these produces a TCA Cycle intermediate or pyruvate # carbons Amino acid alpha-keto acid formed after deamination 3 Alanine (Ala) Pyruvate 4 Aspartate (Asp) Oxaloacetate 5 Contains Glutamate (Glu) Alpha ketoglutarate 1 amino group Glutamine (Gln) Contains 2 amino groups Glutamate acts as acts as –NH3 acceptor (in AA degradation, accepts –NH3) forming glutamine acts as –NH3 donor (for biosynthetic pathways/excretion) forming alpha-ketoglutarate Glutamine & Alanine key transporters of amino groups between tissues and liver. levels of these amino acids in blood is higher than all other amino acids. LO5: Describe how the body degrades amino acids Describe how nitrogen is removed to produce carbon skeletons and the pathways involved  Some detail of deamination Before the carbon skeletons of amino acids can be used they must be deaminated: R Carbon skeleton R O COO- + + H3N COO- NH4+ Amine group (as ammonium ion) 2 key mechanisms for deamination (removing the amino group): 1. Transamination Transfer of amino group to a suitable keto acid acceptor (no free amine released) Enzymes involved are aminotransaminases such as alanine aminotransferase (ALT) or aspartate aminotransferase (AST) Reactions are reversible, enzyme requires pyridoxal phosphate (B6) 2. Oxidative deamination Oxidative removal of a free amino group forming an keto acid + free ammonia via glutamate dehydrogenase (shown in above reaction) LO5: Describe how the body degrades amino acids Describe how nitrogen is removed to produce carbon skeletons and the pathways involved  Catabolism of amino acids 1. Transamination Transfer of amino group from one substrate to another  From an amino acid to a keto acid acceptor (usually -ketoglutarate) Catalyzed (reversibly) by aminotransferase enzymes (transaminases) LO5: Describe how the body degrades amino acids  Examples of Transamination Reactions PLP PLP  α-ketoglutarate is an amine acceptor in the forward direction  Aminotransferase enzymes require the cofactor pyridoxal phosphate (PLP) (derived from vitamin B6, pyridoxine), which helps the enzyme by carrying the amino group during the reaction  There are a range of these enzymes with differential distribution – each specific for particular amino acids.  Alanine aminotransferase (ALT) & aspartate aminotransferase (AST) form part of the liver function suite of blood test Damaged cells release enzymes into the blood. Measurement of their activity from a blood sample indicates cell damage. Determining which isoform is present can help identify the organ involved (liver/muscle etc) LO5: Describe how the body degrades amino acids Appreciate the role of the B-group vitamin pyridoxine (B6) in amino acid metabolism  Catabolism of amino acids 2. Deamination – removal of ammonia Glutamate is the only amino acid that doesn’t have to transfer tis amino group to another molecule It undergoes oxidative deamination - glutamate dehydrogenase removes the amine group and hydrogens Glutamate + NAD+ + H20 ↔ α-Ketoglutarate + NADH + H+ + NH4+ The ammonium produced is used to form urea Thus glutamate dehydrogenase plays a key role in ammonia metabolism LO5: Describe how the body degrades amino acids Describe how nitrogen is removed to produce carbon skeletons and the pathways involved  Catabolism of amino acids 2. Deamination – removal of ammonia LO5: Describe how the body degrades amino acids Describe how nitrogen is removed to produce carbon skeletons and the pathways involved  Catabolism of Amino Acids Cont’d  Catabolism can provide starting material for lipids, energy and glucose  Following deamination the carbon chains of the amino acids are fed into central pathways of metabolism (TCA Cycle, glycolysis etc)  Pathways converge to 7 key intermediates LO5: Describe how the body degrades amino acids Describe how nitrogen is removed to produce carbon skeletons and the pathways involved  Mechanism of Toxicity of Excess Ammonia  Ammonia (NH3) readily crosses the BBB by diffusion so any process that increases serum ammonia is potentially dangerous ie hyperammonaemia (*differentiation between NH3 (neutral base) and its conjugate acid NH 4+ (cationic ammonium) – at physiological pH it is mostly present as the latter - uncharged species cross lipid membranes more easily unless transported)  Proposed mechanisms of ammonium toxicity include (among others):  Increased levels of glutamate (excitatory neurotransmitter/excitotoxin)  Activation of NMDA receptors  Increased levels of glutamine (causing oedema)  Depletion of ATP (interferes with mitochondrial function, possibly through inc. free radicals)  Terminal stages of ammonia toxicity include coma, brain swelling & death  The urea cycle is used to minimise risks of excess ammonia in circulation  Ammonia is produced during catabolism of amino acids, purines & pyrimidines & used up in their synthesis & synthesis of urea. Achieving a balance is crucial to health LO5: Describe how the body degrades amino acids Describe how nitrogen is removed to produce carbon skeletons and the pathways involved  Urea Cycle  High levels of ammonia are toxic and lead to lethargy or NH4+ mental retardation & eventually death if untreated. The liver & kidneys work together to ensure toxic levels of ammonia do not accumulate  Urea Cycle combines 2 amino groups into the urea molecule  Urea is subsequently transported via the blood supply to the kidney where it is excreted, and excreted in sweat O + H 3N NH3+ Urea LO6: Outline the role of the urea cycle in processing ammonia to urea  Overview of the Urea Cycle 1. Ammonia released from glutamate glutamate dehydrogenase reaction is incorporated NH4+ into carbamoyl phosphate -KG HCO3- 1 Carbamoyl phosphate 2. Carbamoyl phosphate combines with ornithine forming citrulline Mitochondrion Cytosol 3. Citrulline combines with Ornithine Citrulline aspartate which donates 2nd amine Urea Aspartate Malate Donates 2nd amine of urea (N) OAA Arginine Argininosuccinate Link to TCA Cycle Fumarate LO6: Outline the role of the urea cycle in processing ammonia to urea Very simple overview of the urea cycle + CO2 + NH3 - H2O + NH3 - H2O  O Formation of Urea + H 3N NH3+ Urea  One amine is derived from NH4+ (produced primarily from glutamate via deamination - the glutamate dehydrogenase reaction)  enters cycle via carbamoyl phosphate  Second amine NH3+ is derived from aspartate (formed by the transamination of the α-keto acid oxaloacetate)  fumarate is formed by this process & recycled via TCA cycle to oxaloacetate LO6: Outline the role of the urea cycle in processing ammonia to urea Sources of Nitrogen for Urea Cycle LO6: Outline the role of the urea cycle in processing ammonia to urea Trace the flow of N atoms, and outline how the cycle overlaps with the TCA cycle  Summary of The Urea Cycle 3 4 2 5 1  The synthesis of urea occurs almost exclusively in the liver  4 reactions – first occurs in the mitochondrial matrix, then citrulline moves into the cytosol, urea is released from arginine and ornithine is regenerated.  The urea formed diffuses into the blood and goes to the kidney  High rates of amino acid breakdown result in elevated glutamate (glutamic acid) concentrations which increases the supply of substrate for the cycle LO6: Outline the role of the urea cycle in processing ammonia to urea  Urea and TCA cycles overlap  The urea cycle is really two cycles (Kreb’s bicycle!)  The carbon flow is split between urea synthesis and the recycling of fumarate to aspartate, this second part occurring in the mitochondria and involving part of the TCA cycle  Urea synthesis however occurs almost exclusively in the liver. LO6: Outline the role of the urea cycle in processing ammonia to urea Connection between urea cycle and TCA cycle  Fumarate, which is formed from the cleavage of arginosuccinate, is an intermediate of the citric acid cycle  Fumarate is converted to malate and then to oxaloacetate (an -keto acid)  Oxaloacetate can acquire a second amino group to become aspartate – returns to the urea cycle LO6: Outline the role of the urea cycle in processing ammonia to urea  Regulation of the urea cycle  Urea Cycle is a waste cycle - usually regulated by a feed forward mechanism in which the rate of the cycle responds to increased ammonia  Urea cycle increases or decreases in response to high/low protein diet  Regulation is at the level of the enzyme that synthesises carbamoyl phosphate (ie. the beginning of the pathway, which avoids waste as this is an anabolic pathway requiring ATP)  In acidosis urea synthesis is decreased and NH4+ excretion is increased to excrete protons  During fasting increased amino acid metabolism to fuel gluconeogenesis increase urea synthesis LO6: Outline the role of the urea cycle in processing ammonia to urea  Urea Cycle Disorders  Genetic disorders resulting from a defect in synthesis/function of one of the urea cycle enzymes  Symptoms arise in infancy (often triggered by switch from human milk to formula or introduction of solid foods – both higher in protein)  Symptoms include hyperammonaemia, lethargy, seizures, vomiting, hypotonia (poor muscle tone), respiratory alkalosis, coma (even death if untreated)  Severity of the condition depends on which enzyme is affected.  Most common is ornithine transcarbamoylase deficiency - severe neonatal symptoms – X-linked inheritance  Blood tests would show increased blood levels of ammonia and/or build up of one or more urea cycle intermediates LO6: Outline the role of the urea cycle in processing ammonia to urea  Clinical Measurement of Urea  Urea is the principal nitrogenous waste product of metabolism and is generated from protein breakdown. It’s one of the main solutes in urine.  Urea is synthesized in the liver via the urea cycle. ~80% of urea is cleared by the kidneys. Decreased urinary excretion of urea is associated with kidney disease.  Blood urea is an important parameter measured in patients with metabolic conditions that affect amino acid metabolism. It can form one of the tests which assess kidney* or liver disease.  Increased blood urea can be caused by  urea production,  urea elimination, or a combination of the two.  serum urea (seen in heart failure, dehydration, or a diet high in protein)  serum urea (seen in liver damage) Assay: Urease is commonly used in assays for this purpose Urea  CO2 + NH3 LO6: Outline the role of the urea cycle in processing ammonia to urea Summary Some questions…… Which organ is primarily responsible for the metabolism of amino acids? A.Liver B.Kidneys C.Stomach D.Brain E.Skeletal muscle Which organ is primarily responsible for the metabolism of amino acids? A.Liver B.Kidneys C.Stomach D.Brain E.Skeletal muscle Answer A. Liver – whilst transamination and ‘remodelling’ of amino acids can occur in most tissues, the liver is the primary site of aa metabolism. What is the first step in the catabolism of most amino acids? A.Transamination B.Phosphorylation C.Decarboxylation D.Oxidative deamination E.Reduction What is the first step in the catabolism of most amino acids? A.Transamination B.Phosphorylation C.Decarboxylation D.Oxidative deamination E.Reduction Answer A. Liver – transamination – transfer of the amino group to a suitable keto acid (no free amine released at this point) Which vitamin is a cofactor for the transamination reactions that interconvert various amino acids, such as alanine and pyruvate? A)Vitamin A B)Vitamin C C)Vitamin B6 (pyridoxine) D)Vitamin D E)Vitamin B12 Which vitamin is a cofactor for the transamination reactions that interconvert various amino acids, such as alanine and pyruvate? A)Vitamin A B)Vitamin C C)Vitamin B6 (pyridoxine) D)Vitamin D E)Vitamin B12 Answer: C) Vitamin B6 (pyridoxine) What is the primary function of the glucose-alanine cycle in the human body? A.Energy production in the liver B.Amino acid synthesis C.Provide substrates for gluconeogenesis D.Lipid metabolism E.Production of urea in the skeletal muscle What is the primary function of the glucose-alanine cycle in the human body? A.Energy production in the liver B.Amino acid synthesis C.Provide substrates for gluconeogenesis D.Lipid metabolism E.Production of urea in the skeletal muscle ANSWER: C) Provide substrates for gluconeogenesis – breakdown of amino acids in the skeletal muscle produces alanine - that is released and taken up by the liver. The amino group is released from alanine (then removed by formation urea in the liver), whilst the carbon skeleton of alanine is used in gluconeogenesis to provide glucose to the plasma. In the urea cycle, what is the primary purpose of converting ammonia to urea? A)To eliminate excess nitrogen from the body B)To produce ATP C)To synthesize amino acids D)To regulate blood pH E) To provide carbon skeletons for amino acid synthesis In the urea cycle, what is the primary purpose of converting ammonia to urea? A)To eliminate excess nitrogen from the body B)To produce ATP C)To synthesize amino acids D)To regulate blood pH E) To provide carbon skeletons for amino acid synthesis Answer: A) To eliminate excess nitrogen from the body (since ammonia is toxic)  Session Learning Objectives LO1: Review the basic structure of amino acids and their roles in the body LO2: Be aware of the classification essential/non-essential and glucogenic/ketogenic amino acids LO3: Outline the biosynthetic pathways for amino acids LO4: Explain the metabolic relationship between amino acid metabolism to central metabolism (eg. TCA cycle) LO5: Describe how the body degrades amino acids Describe how nitrogen is removed to produce carbon skeletons and the pathways involved Appreciate the role of the B-group vitamin pyridoxine (B6) in amino acid metabolism LO6: Outline the role of the urea cycle in processing ammonia to urea Trace the flow of N atoms, and outline how the cycle overlaps with the TCA cycle Useful texts: Baynes and Dominiczak, Chapters 18 and 19