Amino Acid Metabolism 2 PDF

Amino Acid Metabolism 2 Danny Zisterer  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 t...

Amino Acid Metabolism 2 Danny Zisterer  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  The Proteasome Digests the Ubiquitin-Tagged Proteins Proteasome (26S proteasome) = a large, ATP-driven protease complex that digests ubiquitinated proteins The 26S proteosome is a complex of two components: – one 20S catalytic unit arranged as a barrel – two 19S regulatory units that control access to the interior of the 20S catalytic subunit  Functions of the 19S Regulatory Unit The 19S regulatory units: – contain ubiquitin receptors that bind specifically to polyubiquitin chains – use ATP to unfold polyubiquitinated chains and direct them into the catalytic core – contain an isopeptidase that cleaves off intact ubiquitin molecules so that they can be reused Key components of the 19S complex are six ATPases The Proteolytic Active Sites of the 20S Barrel There are three types of active sites in the β subunits, each with a different specificity. All active sites employ an N-terminal Thr residue. – The hydroxyl group of the Thr residue attacks the carbonyl groups of peptide bonds, forming acyl-enzyme intermediates. – Substrates are degraded in a processive manner without intermediate release. – Substrates are reduced to peptides ranging from seven to nine residues before release. The Proteasome and Other Proteases Generate Free Amino Acids  Processes Regulated by Protein Degradation Many biological Gene transcription processes are Cell-cycle progression controlled, at least Organ formation in part, by protein Circadian rhythms degradation via the Inflammatory response ubiquitin- Tumor suppression proteasome Cholesterol metabolism pathway. Antigen processing  Protein Degradation Can Be Used to Regulate Biological Function Bortezomib (Velcade) = a dipeptidyl boronic acid inhibitor of the proteasome – used as a therapy for multiple myeloma HT1171 = inhibitor of the proteasome of M. tuberculosis – has no effect on human proteasomes  The First Step in Amino Acid Degradation Is the Removal of Nitrogen Amino acids not needed as building blocks are degraded to compounds able to enter the metabolic mainstream. The amino group is removed, and then the remaining carbon skeleton is metabolized to a glycolytic intermediate or to acetyl CoA. The major site of amino acid degradation in mammals is the liver. Muscles also readily degrade the branched-chain amino acids (Leu, Ile, and Val). Fate of amino acids in liver and cells carbon skeletons oxidation Amino acids Skeletal muscle Branched chain amino acids (e.g. leucine, isoleucine and valine) are mainly oxidized in skeletal muscle  Alpha-Amino Groups of Many Amino Acids Are Converted into Ammonium Ions by the Oxidative Deamination of Glutamate in the Liver The α-amino group is transferred to α-ketoglutarate, yielding glutamate. Glutamate is oxidatively deaminated in the liver to yield ammonium ion (NH4+).  The Role of Aminotransferases Aminotransferases (transaminases) = catalyze the transfer of an α-amino group from an α-amino acid to an α- ketoacid – Reactions are reversible and can be used to synthesize amino acids from α-ketoacids.  The Role of Glutamate Dehydrogenase Glutamate dehydrogenase = a mitochondrial enzyme that converts the nitrogen atom in glutamate to a free ammonia ion by oxidative deamination – is essentially a liver-specific enzyme – can use either NAD+ or NADP+ – proceeds by dehydrogenation of the C–N bond, followed by hydrolysis of the ketimine – allosterically inhibited by GTP and stimulated by ADP in mammals  The Fate of the Ammonia Ion The sum of the reactions of aminotransferases and glutamate dehydrogenase is In most terrestrial vertebrates, NH4+ is converted into urea, which is excreted.  Serine and Threonine Can Be Directly Deaminated Serine dehydratase and threonine dehydratase directly deaminate their respective amino acids. – PLP is the prosthetic group. Dehydration precedes deamination. Serine → pyruvate + NH4 + Threonine → -ketobutyrate + NH4 +  Protein turnover stats Protein turnover = the degradation and resynthesis of proteins Protein turnover– an index of basal metabolism Regulation of Protein Turnover  Peripheral Tissues Transport Nitrogen to the Liver Muscle uses branched-chain amino acids as fuel during prolonged exercise and fasting. Muscle lacks enzymes of the urea cycle. Nitrogen is transported from muscle to the liver as alanine (through glutamate) in the glucose-alanine cycle. Amino groups from amino acids can also be transferred to glutamine. Glutamine synthetase = catalyzes the synthesis of glutamine from glutamate and NH4+ – Nitrogens of glutamine can be eliminated by incorporation into urea in the liver. NH4 + + glutamate + ATP ⎯⎯⎯⎯⎯⎯⎯ Glutamine synthetase → glutamine + ADP + Pi Pathway Integration: The Glucose–Alanine Cycle Allows Muscle Cells to Use Amino Acids as Fuel During prolonged exercise and fasting, muscle uses branched-chain amino acids as fuel. The nitrogen removed is transferred (through glutamate) to alanine, which is released into the blood stream. In the liver, alanine is taken up and converted into pyruvate for the subsequent synthesis of glucose. How alanine is used to transfer amino groups to the liver -ketoglutarate -ketoglutarate glutamate pyruvate alanine aminotransferase alanine aminotransferase  Ammonium Ions Are Converted into Urea in Most Terrestrial Vertebrates Some of the NH4+ formed in the breakdown of amino acids is consumed in the biosynthesis of nitrogen compounds (e.g. nucleotide bases) Excess NH4+ is converted into urea by the urea cycle (in ureotelic organisms). Urea is a highly water-soluble, inert, non-toxic molecule. Its essential role is to prevent accumulation of the toxic NH4+ ion produced from amino groups. Glutamate dehydrogenase : a major source of NH4+ in liver Where does the NH4+ go?  The Urea Cycle Eliminates Both Nitrogen and Carbon Waste Products The Urea Cycle- straddles the mitochondria and the cytoplasm arginase fumarase Malate (cytoplasmic) Malate dehydrogenase NAD+ NADH2 Glutamate -ketoglutarate Oxaloacetate Transamination: aspartate amino transferase Flux increased by increased (a) protein intake, and (b) glucagon and glucocorticoids (synergistically)  The Urea Cycle Begins with the Formation of Carbamoyl Phosphate Carbamoyl phosphate synthetase I = catalyzes the coupling of ammonia (NH3) with bicarbonate (HCO3–) to form carbamoyl phosphate – occurs in the mitochondria – requires two molecules of ATP – is the key regulatory enzyme for urea synthesis-can be regulated allosterically and by covalent modification  Carbamoyl Phosphate Synthetase I Is the Key Regulatory Enzyme for Urea Synthesis Carbamoyl phosphate synthetase I: – requires the allosteric regulator N-acetylglutamate (NAG) for activity (synthesized by N- acetylglutamate synthase-itself activated by arginine). NAG is made when amino acids (arginine and glutamate) are readily available. – CPSI is maximally activated when amino acids are being metabolized for fuel use – is inhibited by acetylation and stimulated by deacetylation.  The Sirtuin family of Protein Deacetylases- role of SIRT 3,4 and 5 in the Urea cycle Mitochondrial function of sirtuins in amino acid degradation and the urea cycle. Sirt3 deacetylates and activates ornithine transcarbamoylase (OTC); Sirt4 inhibits glutamate dehydrogenase (GDH), which is involved in the synthesis of glutamate and amino acid-induced insulin release, whereas Sirt5 is also involved in the urea cycle through activating carbamoyl-phosphate synthase 1 (CPS1). Sirtuin activity is coupled to metabolism via NAD+ levels, which rise in conditions of energy poor state- nutrient shortage. Because sirtuins require NAD+ for their catalytic activity, their enzymatic activity is higher in situations of energy distress. Control of flux through the Urea Cycle: 1. High protein diet 2. Glucagon increases degradation of amino acids and glucocorticoid increases arginase expression: act synergistically 3. Arginine via N-acetylglutamate allosterically activates CPS1 4. High NAD+/NADH ratio via SIRT5 deacetylates CPS1 activating it 5. High NAD+/NADH ratio via SIRT3 deacetylates OTC activating it 6. High ADP/ATP or GDP/GTP ratio allosterically activates glutamate dehydrogenase to deaminate glutamate Inherited Defects of the Urea Cycle: Humans excrete approximately 10kg of urea per year. Synthesis of urea in the liver -major route for the removal of NH4+ All defects in the urea cycle lead to elevated level of NH4+ in the blood (hyperammonemia). Highly toxic. Some genetic defects become evident and day or two after birth- infant become lethargic and vomits periodically. Coma and irreversible brain damage may follow (hepatic encephalopathy). High levels of NH4+ may: – inappropriately activate an Na+-K+-Cl– cotransporter, disrupting the osmotic balance of the nerve cell and causing cellular swelling. – disrupt neurotransmitter systems. – impact energy metabolism, levels of oxidative stress, nitric oxide synthesis, and signal transduction pathways.  Argininosuccinase Deficiency Can Be Managed by Supplementing the Diet with Arginine Argininosuccinase (also called arginosuccinate lyase) deficiency is treated by: – supplementing the diet with arginine. – restricting total protein intake. Excess nitrogen is excreted in the form of argininosuccinate.  The Urea Cycle Eliminates Both Nitrogen and Carbon Waste Products  Urea Is Not the Only Means of Disposing of Excess Nitrogen Ammoniotelic organisms = organisms that release nitrogen as NH4+ and rely on the aqueous environment for dilution – examples: aquatic vertebrates and invertebrates Uricotelic organisms = secrete excess nitrogen as the purine uric acid – examples: birds and reptiles – requires very little water Summary: The proteasome is a large complex that digests ubiquitinated proteins using ATP. The resulting amino acids provide a source of precursors for proteins, nucleotide bases etc. Amino acids in excess of immediate requirements are deaminated; the amino nitrogen is mainly converted to urea and excreted. The C-H skeletons are oxidised to release energy or converted to fat or glycogen according to metabolic controls at that time and the particular amino acid. Most amino acids are deaminated via transamination with a- ketoglutarate. The glutamate formed is deaminated by glutamate dehydrogenase releasing ammonia. Peripheral tissues transport nitrogen to the liver. Nitrogen is transported from muscle to the liver as alanine (through glutamate) in the glucose-alanine cycle. Amino groups from amino acids can also be transferred to glutamine. In liver, glutamine is hydrolysed by glutaminase to release ammonium ion for urea synthesis. Summary: First step in synthesis of urea is the formation of carbamoyl phosphate-from HCO3-, NH3 and 2ATP. A 2nd reaction in mitochondria produces citrulline, which leaves the mito and condenses with aspartate-which provides the second nitrogen of what will become urea. Two subsequent reactions in cytosol produce urea and regenerate intermediates of cycle. There are a number of inherited defects of the urea cycle (prevalence of 1 in 15,000) that cause hyperammonemia and can lead to brain damage. 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)