Amino Acid Metabolism 1 - Danny Zisterer PDF

Amino Acid Metabolism 1 Danny Zisterer  Amino Acid Metabolism & Protein Turnover The digestion of proteins from the diet and the degradation of proteins within the cell provides a steady supply of amino acids In response to metabolic demands, cellular proteins are broken down and re-synthes...

Amino Acid Metabolism 1 Danny Zisterer  Amino Acid Metabolism & Protein Turnover The digestion of proteins from the diet and the degradation of proteins within the cell provides a steady supply of amino acids In response to metabolic demands, cellular proteins are broken down and re-synthesised Primary use of amino acids-building blocks for synthesis of proteins and other nitrogenous compounds such as nucleotide bases, heme and neurotransmitters Amino acids in excess of those needed for biosynthesis are not stockpiled for use later-are used as metabolic fuel. After immediate needs are satisfied excess amino acids are oxidised or converted to glycogen or fat  Amino Acid Metabolism & Protein Turnover A study of amino acid metabolism demonstrates the critical connections between basic biochemistry and clinical medicine Virtually all human proteins contain all 20 amino acids. If even one is deficient in the required amount, important functional proteins cannot be made except by destruction of other functional proteins (muscle proteins and haemoglobin) to supply it Extreme forms of malnutrition-insufficient intake of protein Kwashiokor: Leads to muscle wasting, apathy, inadequate growth, brain effects and lowered levels of serum proteins such as albumin. This in turn leads to reduction in osmotic pressure of blood-leads to oedema of tissues Learning Objectives: 1. How nitrogen is incorporated into amino acid/protein 2. How nitrogen is transported around the body 3. What controls protein turnover 4. How the body gets rid of excess nitrogen 5. Identify metabolic errors in amino acid metabolism  The Biosynthesis of Amino Acids Ammonia is the source of nitrogen in all amino acids. Carbon backbones comes from glycolytic pathway, pentose phosphate pathway, or the citric acid cycle.  Biosynthesis of Amino Acids Nitrogen fixation = a process that reduces N2 to NH3 (ammonia)  NITROGEN FIXATION 1. Amino acids, purine, pyrimidines and many other biomolecules contain Nitrogen 2. Nitrogen comprises 80% of the atmosphere and exists as N2 3. N2 (N N) is very inert. Triple bond energy 225kcal mol-1 Combining N with H to make NH3 typically requires 5000C and 300 atm (devised by Fritz Haber in 1910-still used in fertilizer factories) 4. Soil bacteria (Klebseilla, Azotobacter,cyanobacteria) and symbiotic bacteria (Rhizobium) invade the root nodules of leguminous plants and can fix nitrogen. Higher organisms are unable to fix nitrogen. 5. Nitrogen is fixed by the nitrogenase complex which consists of a reductase and iron-molybdenum-containing nitrogenase [@100C and 1atm (100kPa)]  Nitrogen Fixation in Rhizobium Reductase provides electrons with high reducing power and nitrogenase uses the electrons to reduce N2 to NH3  NITROGEN FIXATION 6. At least 16 ATP molecules are hydrolysed to form two molecules of NH3 7. Diazatrophic microrganisms fix 1011Kg per year, 60% of the earths newly fixed nitrogen 8. Ammonia can also be obtained from reduction of the nitrate ion (NO3-)  Maintaining Low Oxygen Concentration in Root Nodules Oxidative phosphorylation, which supplies ATP for nitrogen fixation, requires O2. The nitrogenase complex is extremely sensitive to inactivation by O2. Nitrogenase is located in the root nodules of leguminous plants. Leguminous plants maintain a very low concentration of free O2 in their root nodules, using an O2-binding protein that is a hemoglobin homolog (leghemoglobin). – allows simultaneous functioning of ATP synthesis and nitrogenase Ammonium Ion Is Assimilated into an Amino Acid through Glutamate and Glutamine NH3 generated by the nitrogenase complex becomes NH4+ in aqueous solutions. Glu and Gln act as nitrogen donors for most amino acids. – Glu contributes its α-amino group by transamination in the synthesis of most amino acids. – Gln contributes its side-chain nitrogen atom in the synthesis of Trp and His. Ammonium ion is assimilated into amino acids through glutamate and glutamine-these act as nitrogen donors for most amino acids glutamate dehydrogenase: reductive amination glutamine synthase: a second ammonium ion is incorporated into glutamate to form glutamine reductive amidation  Amino Acids Are Made from Intermediates of the Citric Acid Cycle and Other Major Pathways The majority of amino acids other than Glu and Gln obtain their nitrogen from Glu or Gln. Carbon skeletons for amino acid synthesis are provided by intermediates of glycolysis, the citric acid cycle, or the pentose phosphate pathway.  Amino Acids Can Be Sorted into Biosynthetic Families citric acid cycle glycolysis pentose phosphate pathway pentose phosphate pathway citric acid cycle glycolysis Majority of amino acids obtain nitrogen from glutamate or glutamine. The C skeletons come from intermediates of glycolysis, pentose phosphate pathway or citric acid cycle. AAs that give rise to other AAs shaded in yellow. Essential AAs are in bold.  Humans Can Synthesize Some Amino Acids but Must Obtain Others from Their Diet Essential amino acids = 9 amino acids that humans cannot synthesize and must be supplied in the diet Nonessential amino acids = 11 amino acids that humans can synthesize if dietary content is insufficient Tyr is sometimes designated as essential, but it can be synthesized from available Phe in one step  Basic Set of 20 Amino Acids Nonessential Essential Alanine Histidine Arginine Isoleucine Asparagine Leucine Aspartate Lysine Cysteine Methionine Glutamate Phenylalanine Glutamine Threonine Glycine Tryptophan Proline Valine Serine Tyrosine Nonessential amino acids can be synthesised if dietary content is insufficient Essential and Nonessential Amino Acids Can Be Distinguished by the Required Number of Biosynthetic Steps Those amino acids requiring a large number of steps for their synthesis are essential in the diet because some of the enzymes for these steps have been lost in the course of evolution.  Biosynthesis of Aspartate, Alanine, and Glutamate Formed by the Addition of an Amino Group to an Alpha- Ketoacid. α-Ketoglutarate can be converted into glutamate by reductive amination. Aspartate and alanine can be made from the addition of an amino group to oxaloacetate and pyruvate, respectively. These transamination reactions are catalyzed by pyridoxal phosphate-dependent aminotransferases. Aminotransferases or transaminases catalyse the interconversion of amino acids and alpha-keto acids (e.g. pyruvate, a-ketoglutarate, oxaloacetate) by transfer of amino groups. Aspartate, alanine and glutamate are formed by addition of an amino group to an alpha-keto acids Aspartate aminotransferase catalyses the interconversion of oxaloacetate and glutamate to aspartate and α-ketoglutarate: Oxaloacetate + glutamate (Glu)↔Aspartate (Asp) + α- ketoglutarate Whereas alanine aminotransferase interconverts pyruvate and glutamate to alanine and α-ketoglutarate: Pyruvate + glutamate (Glu)↔Alanine (Ala) + α- ketoglutarate The amino group transfer catalysed by these enzymes are crucial in both amino acid biosynthesis and degradation. Glutamate always serves as one of the amino acids in aminotransferase reactions and is thus the ‘gateway’ between amino groups of most amino acids and free ammonia. Aminotransferases move nitrogen around to an a-ketoacid a-ketoacid a-ketoglutarate (a-ketoglutarate) (a-ketoacid) (a-ketoacid) (a-ketoglutarate) a-keto a-keto a-keto Aspartate aminotransferase (AST) and alanine aminotransferase (ALT), are the two aminotransferases of greatest clinical significance. The primary clinical application of serum AST and ALT measurement is the detection and diagnosis of liver disease. Hepatic cell injury is manifested by elevated serum aminotransferase activity (leakage into blood due to membrane damage) prior to the appearance of clinical symptoms and signs (such as jaundice). Liver damage can occur due to: – viral hepatitis. – long-term excessive alcohol consumption. – reaction to drugs (e.g., acetaminophen). Aminotransferases require the cofactor pyridoxal phosphate  Protein Turnover and Amino Acid Catabolism Some plants have adapted to live in nitrogen-depleted soil by capturing and digesting insects.  Protein Digestion and Turnover Amino acids are obtained from the diet when proteins are digested. Cellular proteins are degraded to amino acids because of damage, misfolding, or changing metabolic demands. Excess amino acids cannot be stored or excreted, so they must be used as metabolic fuel.  Proteins Are Degraded to Amino Acids Dietary proteins are degraded to amino acids, which are absorbed by the intestine and transported in the blood. Essential amino acids = amino acids that cannot be synthesized and must be acquired in the diet. Absorption of Amino Acids in mammals/humans Amino acids are taken to the liver by the hepatic portal vein  Cellular Proteins Are Degraded at Different Rates Protein turnover = the degradation and resynthesis of proteins – takes place constantly in cells – essential for removing short-lived or damaged proteins The half-lives of proteins range over several orders of magnitude – Insulin is approx. 1 hour – Haemoglobin is the life of the red blood cell – The lens protein crystallin is the life of the organism Known long lived proteins and molecules  Role of Ubiquitin in Protein Turnover Ubiquitin = a small (76 aa) protein that tags proteins for destruction – present in all eukaryotic cells – highly conserved Ubiquitin attaches by its carboxyl- terminal Gly residue to the ε-amino groups of 1+ Lys residues on the target protein – requires ATP hydrolysis  Three Enzymes Participate in the Attachment of Ubiquitin to a Protein Ubiquitin-activating enzyme (E1) = adenylates ubiquitin and transfers it to a sulfhydryl group of a Cys residue of E1 – requires ATP Ubiquitin-conjugating enzyme (E2) = transfers ubiquitin to one of its own sulfhydryl groups Ubiquitin–protein ligase (E3) = transfers ubiquitin from E2 to an ε- amino group on the target protein – brings E2 and the target protein together – ubiquitin be transferred directly or be passed to a Cys residue of E3 first  Ubiquitin Conjugation Requires Three Enzymes A chain of 4+ ubiquitin molecules is an especially effective signal for protein degradation.  Importance of E3 Proteins to Normal Cell Function Proteins that are not degraded because of a defective E3 may accumulate, causing a disease of protein aggregation. Angelman syndrome = a severe neurological disorder characterized by cognitive disability, absence of speech, uncoordinated movement, and hyperactivity – caused a defect in a member of the E3 family Overexpression of an E3 linked to autism. Inappropriate protein turnover can lead to cancer. Summary: Amino acids are supplied in diet from protein hydrolysis. Proteins are also constantly degraded and re-synthesised-tightly regulated and requires complex enzyme systems. The body synthesises 11 amino acids-rest obtained by diet. All 20 needed for protein synthesis. In the biosynthesis of amino acids microorganisms use ATP and a powerful reductant to reduce atmospheric nitrogen to ammonia (nitrogen fixation). Higher organisms consume the fixed nitrogen to synthesise amino acids, nucleotides and other nitrogen containing biomolecules. The major points of entry of NH4+ ion into metabolism are glutamate and glutamine-these act as nitrogen donors for most amino acids. Carbon skeletons for amino acid synthesis are provided by intermediates of glycolysis, the citric acid cycle, or the pentose phosphate pathway. Cellular proteins are degraded at widely variable rates-from minutes to life of the organism. Proteins to be degraded are tagged with ubiquitin. References: Textbook of Biochemistry with clinical correlations 7th edition (2010) Thomas M. Devlin (Ed.) Wiley Press Biochemistry (2012) seventh edition, Jeremy M. Berg, John L. Tymoczko, Lubert Stryer, Freeman Press Principles of Biochemistry by David L. Nelson, Albert Lehninger and Michael M. Cox (2008), Fifth edition Freeman Press Metabolic Regulation; A human perspective Keith Frayn 2nd (2003) Blackwell Basic Clinical Biochemistry: A Clinical Approach Dawn B. Marks, Allan D. Marks, John Lieberman 2nd edition (2008)