lecture22a_11_20_08 - Lecture 22a Overview: • proteins...

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Unformatted text preview: Lecture 22a Overview: • proteins are degraded to amino acids • protein turnover is tightly regulated • removal of nitrogen • urea • production of metabolic intermediates HW: 23.2, 23.3, 23.6, 23.7, 23.9, review previous HW problems involving PLP (handout) Chapter 23: Protein Turnover and Amino Acid Catabolism Amino acid catabolism in mammals Degradation of proteins to amino acids Unneeded proteins are degraded. Misfolded or damaged proteins are degraded. They are marked for destruction by covalent attachment to ubiquitin. Proteins are digested and degraded by specific peptidases. Amino acids and di- and tripeptides are absorbed into the intestinal cells through specific transporters. Free amino acids are released into the blood. Digestion of proteins and protein degradation provide steady supply of amino acids to the cell. Cellular proteins are degraded and resynthesized in response to the changing metabolic needs of the cell. Essential amino acids cannot be synthesized and are only acquired from the diet. Protein turnover is tightly regulated Ubiquitin (8.5 kDa) tags proteins for destruction. This protein is highly conserved in eukaryotes. It becomes attached to the !-amino group of lysine residues. Ubiquitin conjugation Ubiquitin-activating enzyme E1 adenylates ubitquitin (Ub), then transfers Ub to one of its own Cys residues. Ub is then transferred to a Cys residue of ubiquitin-conjugating enzyme E2. Finally, the ubiquitin-protein ligase E3 transfers Ub to a Lys residues of the target protein. The proteasome: the executioner Once the protein is tagged by Ub, the proteasome (26S proteasome; composed of a 20S catalytic unit and 19S regulatory unit) carries out the destruction of the protein. 20S catalytic unit: 28 homologous subunits (" and #); four rings of 7 subunits each. Some # subunits include a protease active site at their amino termini. 20S is capped by 19S. The proteasome and other proteases generate free amino acids Ub-modified proteins are processed by the proteasome into peptide fragments and Ub is recycled. mass 700 kDa Amino acid degradation: the first step is removal of nitrogen Degradation of surplus amino acids provides metabolic intermediates for other pathways. The amino group is removed and products are converted to glucose, CAC intermediates, or acetyl CoA. The amino group is transferred to !-ketoglutarate to form glutamate, which is then oxidatively deaminated. Oxidative deamination by glutamate dehydrogenase recall: ! -ketoglutarate is an intermediate in CAC Sum: !-amino acid + NAD+ (or NADP+) !-ketoacid + NH4+ + NADH + H+ (or NADPH) Aminotransferases (or transminases) catalyze the transfer of !-amino groups from !-amino acids to !-ketoacids Recall: pyridoxal phosphate (PLP); derived from Vit B6 PLP is the key coenzyme in all amino acid transformations. PLP enzymes catalyze a wide array of reactions 1. 2. 3. 4. 5. Transamination Decarboxylation Deamination Racemization Aldol cleavage Aspartate aminotransferase Reaction steps: • Schiff-base formation with amino acid substrate and PLP • Protonated form of PLP acts as an 'electron sink' to stabilize intermediates (electrons are attracted to the positive charge on the nitrogen) • The product Schiff base is cleaved at the completion of the reaction. PLP mechanism PLP mechanism Serine deamination leads to pyruvate dehydratase (dehydration/deamination) The urea cycle: how a biological system gets rid of excess nitrogen Formation of urea serine -> pyruvate + NH4+ threonine -> !-ketobutyrate + NH4+ NH4+ + glutamate + ATP glutamine + ADP + Pi (transport of ammonia) glutamine synthetase Urea cycle and reactions that feed amino groups into the cycle The urea cycle consists of four major steps: 1) formation of citrulline from ornithine and carbamoyl phosphate; 2) formation of argininosuccinate through citrullyl-AMP intermediate; 3) formation of arginine from argininosuccinate and release of fumarate; 4) formation of urea and regeneration of ornithine. Formation of urea enzyme: carbamoyl phosphate synthetase couple ammonia with bicarbonate with consumption of 2 ATP Formation of urea Step one Ornithine and citrulline are amino acids, but not used in protein synthesis. anhydride has high transfer potential (of carbamoyl group) Formation of urea Step three glucose synthesis Step two 2nd amino donor ornithine is recycled for another round Step four Formation of urea Fates of the carbon skeletons of amino acids CO2 + NH4+ + 3 ATP + aspartate + 2 H2O urea + 2 ADP + Pi + AMP + PPi + fumarate The carbon atoms of degraded amino acids are converted into pyruvate, acetyl CoA, acetoacetate, or other intermediates in CAC. ...
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