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FCH 532 Lecture 21

FCH 532 Lecture 21. Chapter 32: Translation Quiz today (Wed.) on amino acids Quiz on Friday TCA cycle Quiz on Monday Transamination mechanism. Page 987. Figure 32-43 Some translational initiation (Shine-Dalgarno) sequences recognized by E. coli ribosomes. Page 1321.

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FCH 532 Lecture 21

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  1. FCH 532 Lecture 21 Chapter 32: Translation Quiz today (Wed.) on amino acids Quiz on Friday TCA cycle Quiz on Monday Transamination mechanism.

  2. Page 987

  3. Figure 32-43 Some translational initiation (Shine-Dalgarno) sequences recognized by E. coli ribosomes. Page 1321 Shine-Dalgarno sequences typically start 10-15 nt upstream of the initiation codon. Are only found in prokaryotes.

  4. Figure 32-45 Translational initiation pathway in E. coli. • 50S and 30S associated. • IF3 binds to 30S, causes release of 50S. • mRNA, IF2-GTP (ternary complex), fMet-tRNA and IF1 bind 30S. • IF1 and IF2 are released followed by binding of 50S. • IF2 hydrolyzes GTP and poises fMet tRNA in the P site. Page 1323

  5. Page 1327

  6. Defining tRNA Binding Sites in Different functional States GENERATE RIBOSOMES IN THE FOLLOWING STATES: A (Aminoacyl): EF-Tu.GTP dependent; mRNA dependent; occupied P Site P (Peptidyl): Reactive with Puromycin (Pm) E (Exit): Deacylated tRNA MONITOR BY CHEMICAL FOOTPRINTING: 30S A site protections: 50S P site protections (also X-linkers, EDTA-FeII) Looking at footprint pre and post peptide bond, translocation The data didn't fit into a simple 2 site model HYBRID STATES HAD TO BE INVOKED

  7. tRNA movement occurs independently on 2 subunits via 6 hybrid states. 1. A/T --> 2. A/A --> 3. A/P --> 4. P/P --> 5. P/E --> 6. E In this model the tRNA would "ratchet" its way through the ribosome undergoing 50° rotations along its longitudinal axis from A to P. This model has received support from EM and X-ray studies.

  8. cryo-EM

  9. Aminoacyl-tRNA EF-Tu-GTP EF-Ts GTP EF-Tu-EF-Ts EF-Ts GDP Page 1333

  10. RF-1 = UAA RF-2 = UAA and UGA Cannot bind if EF-G is present. RF-3-GTP binds to RF1 after the release of the polypeptide. Hydrolysis of GTP on RF-3 facilitates the release of RF-1 (or RF-2). EF-G-GTP and ribosomal recycling factor (RRF)-bind to A site. Release of GDP-RF-3 EF-G hydrolyzes GTP -RRF moves to the P site to displace the tRNA. RRF and EF-G-GDP are released yielding inactive 70S Page 1335

  11. Translation • Shine-Dalgarno sequence • Initiation • Elongation • Release

  12. Page 1322

  13. Figure 32-61 Ribosome-catalyzed hydrolysis of peptidyl–tRNA to form a polypeptide and free tRNA. Page 1336

  14. Urea Cycle • Excess nitrogen is excreted after the metabolic breakdown of amino acids in one of three forms: • Aquatic animals are ammonotelic (release NH3 directly). • If water is less plentiful, NH3 is converted to less toxic products, urea and uric acid. • Terrestrial vertebrates are ureotelic (excrete urea) • Birds and reptiles are uricotelic (excrete uric acid) • Urea is made by enzymes urea cycle in the liver. • The overall reaction is: NH3+ NH3 + HCO3-+ -OOC-CH2-CH-COO- Asp 3ATP 2ADP + 2Pi + AMP + PPi O NH2-C-NH2+ -OOC-CH=CH-COO- Fumarate Urea

  15. Urea Cycle • 2 urea nitrogen atoms come from ammonia and aspartate. • Carbon atom comes from bicarbonate. • 5 enzymatic reactions used, 2 in the mitochondria and 3 in the cytosol. NH3+ NH3 + HCO3-+ -OOC-CH2-CH-COO- Asp 3ATP 2ADP + 2Pi + AMP + PPi O NH2-C-NH2+ -OOC-CH=CH-COO- Fumarate Urea

  16. Page 992

  17. O 2ATP + NH3 + HCO3- NH2-C-OPO3- + 2ADP + 2Pi Carbamoyl phosphate Carbamoyl phosphate synthetase • Carbamoyl phosphate synthetase (CPS) catalyzes the condensation and activation NH3 and HCO3- to form carbomyl phosphate (first nitrogen containing substrate). • Uses 2 ATPs. • Eukaryotes have 2 types of CPS enzymes • Mitochondrial CPSI uses NH3 as its nitrogen donor and participates in urea biosynthesis. • Cytosolic CPSII uses glutamine as its nitrogen donor and is involved in pyrimidine biosynthesis.

  18. Figure 26-8 The mechanism of action of CPS I. • CPSI reaction has 3 steps • Activation of HCO3- by ATP to form carboxyphosphate and ADP. • Nucelophilic attack of NH3 on carboxyphosphate, displacing the phsophate to form carbamate and Pi. • Phosphorylation of carbamate by the second ATP to form carbamoyl phosphate and ADP The reaction is irreversible. Allosterically activated by N-acetylglutamate. Page 993

  19. Figure 26-9 X-Ray structure of E. coli carbamoyl phosphate synthetase (CPS). • E. coli has only one CPS (homology to CPS I and CPS II) • Heterodimer (inactive). • Allosterically activated by ornithine (heterotetramer of (4). • Small subunit hydrolyzes Gln and delivers NH3 to large subunit. • Channels intermediate of two reactions from one active site to the other. Page 993

  20. Page 992

  21. Ornithine transcarbomylase • Transfers the carbomoyl group of carbomyl phosphate to ornithine to make citrulline • Reaction occurs in mitochondrion. • Ornithine produced in the cytosol enters via a specific transport system. • Citrulline is exported from the mitochondria.

  22. Page 992

  23. Arginocuccinate Synthetase • 2nd N in urea is incorporated in the 3rd reaction of the urea cycle. • Condensation reaction with citrulline’s ureido group with an Asp amino group catalyzed by arginosuccinate synthetase. • Ureido oxygen is activated as a leaving group through the formation of a citrulyl-AMP intermediate. • This is displaced by the Asp amino group to form arginosuccinate.

  24. Figure 26-10 The mechanism of action of argininosuccinate synthetase. Page 994

  25. Arigininosuccinase and Arginase • Argininosuccinse catalyzes the elimination of Arg from the the Asp carbon skeleton to form fumurate. • Arginine is the immediate precursor to urea. • Fumurate is converted by fumarase and malate dehydrogenase to to form OAA for gluconeogenesis. • Arginase catalyzes the fifth and final reaction of the urea cycle. • Arginine is hydrolyzed to form urea and regenerate ornithine. • Ornithine is returned to the mitochondria.

  26. Carbamoyl phosphate synthetase (CPS) Ornithine transcarbamoylase Argininosuccinate synthetase Arginosuccinase Arginase Page 992

  27. Regulation of the urea cycle • Carbamoyl phosphate synthetase I is allosterically activated by N-acetylglutamate. • N-acetylglutamate is synthesized from glutamate and acetyl-CoA by N-acetylglutamate synthase, it is hydrolyzed by a specific hydrolase. • Rate of urea production is dependent on [N-acetylglutamate]. • When aa breakdown rates increase, excess nitrogen must be excreted. This results in increase in Glu through transamination reactions. • Excess Glu causes an increase in N-acetylglutamate which stimulates CPS I causing increases in urea cycle.

  28. Metabolic breakdown of amino acids • Degradation of amino acids converts the to TCA cycle intermediates or precursors to be metabolized to CO2, H2O, or for use in gluconeogenesis. • Aminoacids are glucogenic, ketogenic or both. • Glucogenic amino acids-carbon skeletons are broken down to pyruvate, -ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate (glucose precursors). • Ketogenic amino acids, are broken down to acetyl-CoA or acetoacetate and therefore can be converted to fatty acids or ketone bodies.

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