2024-25 Post-Absorption Processing of Protein PDF

POST- ABSORPTION PROCESSING OF PROTEIN DR. STEPHEN KEELY YEAR 2 – GIHEP MODULE LEARNING OUTCOMES Describe the functions of amino acids in the body Discuss the synthesis non-essential amino-acids (in particular the role played by transaminase enzymes) Understand the role of amino acids a...

POST- ABSORPTION PROCESSING OF PROTEIN DR. STEPHEN KEELY YEAR 2 – GIHEP MODULE LEARNING OUTCOMES Describe the functions of amino acids in the body Discuss the synthesis non-essential amino-acids (in particular the role played by transaminase enzymes) Understand the role of amino acids as substrates in gluconeogenesis, ketogenesis and in energy-yielding metabolism Describe ureogenesis and the elimination of amino nitrogen from the body Describe a pathophysiological example of a Urea Cycle disorder [e.g.: OTC Deficiency; OMIM #311250] Amino-acids Amino acids are made up of carbon skeletons linked to an amino group Classification Amino Carbon group skeleton *Conditionally essential Amino-acids (i) ‘Building blocks’ of protein (ii) Precursors of biologically-active compounds nucleic acids – purines and pyrimidines aspartate, glycine & glutamine; aspartate & glutamine haem glycine hormones thyroxine neurotransmitters dopamine, catecholamines biologically-active peptides gonadotropins, PTH, insulin iii) Energy production Sources of Amino-acids Circulating amino-acids come from either 1. The diet 2. Protein turnover Diet Protein Turnover Converted to other amino acids Energy production ~ 300g of body proteins are turned over daily, - mainly muscle proteins Protein Turnover Protein half-life Most proteins T1/2 approx. 2-3 days e.g. Cytochromes Some enzymes T1/2 ≥ 30 minutes e.g. HMG-CoA Reductase Control of protein degradation [a] N-terminal residues determine protein half-life e.g. Ser  T1/2 >20 hr. Asp  T1/2 ~ 3 min. [b] Rapidly degraded proteins have ‘PEST’ amino-acid sequences Pro (P); Glu (E); Ser (S); Thr (T) PEST is a signal for degradation by Lysosomal peptidases Protein degradation to amino acids Lysosomal Degradation - proteins are endocytosed and trafficked to lysosomes for digestion by proteases The Ubiquitin – Proteasome system - proteins are tagged with ubiquitin and sent to proteasomes for degradation Proteasomes = cytosolic protein complexes that degrade proteins by proteolysis (a chemical reaction that breaks peptide bonds). Summary of Amino Acid Turnover - Amino-acids are not stored - re-used in protein synthesis - used for biosynthetic products - metabolised for energy  The First Step in Amino Acid Metabolism is Deamination - Occurs by a process known as transamination - Catalysed by enzymes known as transaminases Transaminases (aminotransferases) are enzymes that catalyse a transamination reaction between an amino acid and an a-keto acid - involved in the deamination of all amino acids (except threonine, lysine & proline) Transaminases  Transamination The most common acceptor of amino groups during AA transamination is a-ketoglutarate Liver Glutamate then undergoes oxidative deamination by the enzyme, Produces a new a-keto acid glutamate dehydrogenase, to recycle for energy metabolism and a-ketoglutarate and release NH3 glutamate  Metabolism Transamination Excretion as Urea  Transaminases - Transaminases are among most abundant enzymes in human tissues - Found in the cytosol in liver, kidney, skeletal and cardiac muscle. - Use vitamin B6 (pyridoxal-phosphate) as a co-factor - Two most important are ALT and AST Catalyses transfer of amino Catalyses transfer of amino group from alanine to a-KG group from aspartate to a-KG - Levels of ALT and AST can be used to diagnose liver (and other diseases) - Since ALT is expressed at higher levels in liver than other tissues, increased levels of this enzyme are more indicative of liver disease  a-Keto Acids a-keto acid a-keto acids are intermediates of the TCA cycle - can be used to generate ATP or can create energy stores through gluconeogenesis - can also be used to synthesise amino acids and therefore provide a mechanism for interconversion of AAs as required by the body  Biosynthesis of Non-Essential Amino Acids Glycolytic and TCA cycle intermediates generate the nonessential amino acids - 3-phosphoglycerate generates serine (serine can then be converted into glycine or cysteine) - α-ketoglutarate can be converted into multiple amino acids, including - glutamate - aspartate - arginine (via the urea cycle) - glutamine (via glutamine synthase) - proline - Pyruvate can be converted into alanine - Oxaloacetate can be converted into aspartate (which can be converted to asparagine)  Degradation of Amino Acids for Energy - If they are not required for biosynthesis, AAs are used to generate/store energy AAs cannot be directly stored – must first be metabolised to glucose or ketones - The fate of the carbon skeletons of the amino acids depends on the physiological state of the individual - Fasting state – AAs can enter the TCA cycle after metabolism to keto acids or acetyl Co-A - Fed State – AAs can be converted to glucose and stored as glycogen or ketone bodies  Degradation of Amino Acids for Energy Amino acids are classified as glucogenic, ketogenic, or both based on which of the seven intermediates produced during their catabolism. Glucogenic Amino acids that can be converted into glucose through gluconeogenesis = AAs which yield pyruvate or TCA cycle intermediates Ketogenic Amino acids that can be converted into ketone bodies by ketogenesis = AAs whose catabolism yields either acetoacetate or its precursors, (acetyl CoA or acetoacetyl CoA) Some amino acids are both glucogenic or ketogenic  Degradation of Glucogenic Amino Acids for Energy Fasting State - Glucogenic amino acids enter the TCA cycle to provide energy NADH FADH Oxidative phosphorylation ETC ATP  Storage of Amino Acids as Glycogen Fed state - Glucogenic amino acids are converted to glucose and stored as glycogen Glycogen Glycogen synthase Glycolysis Gluconeogenesis Amino Acids  Maintenance of plasma glucose levels by Amino Acids Since AAs can undergo gluconeogenesis they play an important role in maintenance of plasma glucose Two principal organs involved are: Muscle - generates >50% of circulating amino-acids Liver - uses amino acids in gluconeogenesis The primary pathway for transfer of amino acids from muscle to liver cells is the Glucose/Alanine Cycle (Cahill Cycle)  Glucose/Alanine Cycle - During fasting or intense exercise muscle proteins can be broken down into AAs - AAs undergo deamination - Keto acids used for energy - NH4 is transferred to glutamate Glutamate then undergoes transamination with pyruvate to form alanine - carried out by alanine transaminase (ALT) The liver converts the carbon skeleton (ie., pyruvate) of Alanine to Glucose by gluconeogenesis  Degradation of Ketogenic Amino Acids for Energy - Ketogenic amino acids are converted to ketone bodies - Occurs either by i) direct conversion to acetoacetate ii) Conversion to precursors of acetoacetate - ketone bodies then travel in the blood to provide energy to other tissues (particularly the brain) Ketolysis (Heart, Brain, Muscle) b-hydroxybutyric acid  SUMMARY - Circulating AAs cannot be stored but can be used to synthesise new proteins & other biomolecules (e.g., nucleic acids, Haem) or to generate energy (ATP) - Classified as glucogenenic or ketogeneic - Important step in metabolism of glucogenic amino acids is transamination - carried out by transaminases (e.g, ALT, AST) and generates - Carbon skeleton (a-keto acids) - NH4+ (ammonium) - The fate of a-keto acids depends on metabolic state: - converted to non-essential amino acids - enter glycolysis and the TCA cycle to produce energy - undergo gluconeogenesis to store energy - Ketogenic amino acids generate acetyl CoA and ketone bodies (provide energy to muscle, heart & brain) - NH4+ released from AAs – toxic & eliminated as urea What about the ammonia?? - Amino-acids are not stored - re-used in protein synthesis - used for biosynthetic products - metabolised for energy  Overview of amino acid catabolism e.g. AST aspartate oxaloacetate 3 key stages: ALT alanine pyruvate 1)Transamination = transfer of amino group to alpha-ketoglutarate by 1 (throughout body) transaminases (generates an alpha-keto acid and glutamate) 2) Oxidative deamination of 2 glutamate by glutamate dehydrogenase (mainly in liver) (generates ammonia + a-ketoglutarate) 3) Urea cycle (converts toxic NH3 to 3 urea) (in liver) For reading see: Harpers Ch28 Meisenberg Ch 26  Transport of Ammonium Three amino acids are important in the transfer of NH3 to the liver 1. Glutamate – formed by transamination of AAs & undergoes oxidative deamination by glutamate dehydrogenase in the liver 2. Glutamine – formed in tissues from glutamate by glutamine synthase - reconverted to glutamate in the liver by glutaminase (releases NH4+) - glutamate undergoes oxidative deamination (releases NH4+) NH4+ NH4+ Glutaminase Glutamate Dehydrogenase a-KG 3. Alanine – main carrier of amino groups from muscle to liver (c.f. glucose/alanine cycle) - ALT transfers amino group to a-KG to give glutamate - glutamate undergoes oxidative deamination (releases NH4+) Amino Acid Degradation: - The Urea Cycle - CONVERTS TOXIC NH3 TO A SAFE FORM – UREA – FOR EXCRETION FROM THE BODY Urea (carbonyl diamide) Urea synthesis takes place in the liver; 5 enzymes; Reactions in cytosol and mitochondria. 2 molecules of ammonia (derived from glutamate & aspartate) are combined with CO2 to form the urea molecule Summary of Amino Acid Catabolism and Urea Production Body Liver Protein Intake Urea Excretion (12-20g N)  Hyperammonaemia Levels of free ammonia in the blood serum are normally 5 – 50 μmol/L Hyperammonaemia occurs if levels exceed the Urea Cycle capacity (> 1,000 μmol/L) Hereditary hyperammonaemia - caused by an inherited Urea Cycle defect (IEMs) (e.g., ornithine transcarbamylase deficiency) Acquired hyperammonaemia - caused by diseases that cause liver failure (e.g., hepatitis, cirrhosis, drug hepatoxicity) Symptoms: Primarily due to CNS toxicity of NH3 - Tremors, slurred speech, vomiting, cerebral oedema, Coma, Death Treatment: i) Limit intake of ammonia (low protein diet) ii) Increase ammonia excretion (e.g., sodium phenylbutyrate or sodium benzoate) - serve as alternatives to urea for excretion of NH3 iii) Severe cases treated by haemodialysis  ORNITHINE TRANSCARBAMOLYASE DEFICIENCY the most common urea cycle disorder. OTC - converts carbamoyl phosphate and ornithine into citrulline. X-linked disorder - Affects males > females X Pathophysiology: lack of OTC leads to excessive ammonia in the blood (ie., hyperammonemia), - Excess ammonia is toxic to the CNS Symptoms headaches, nausea, vomiting, lethargy, confusion, encephalopathy, coma & death Treatment: low protein diet & nitrogen-scavenging agents (e.g., sodium benzoate). Liver transplant is the only option for a cure  RESOURCES Amino Acid Metabolism https://slideplayer.com/slide/4651310/ Transamination of Amino Acids https://www.youtube.com/watch?v=L4cJ8uq31kY Amino Acid Synthesis & Urea Cycle https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015690/#:~:text =The%20major%20TCA%20cycle%20intermediate,alanine%2C %20aspartate%2C%20and%20arginine. Alanine/Glucose cycle https://www.youtube.com/watch?v=V9CSKTFyaYw Biosynthesis of non-essential amino-acids by transamination a-KG Intermediary metabolism