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FCH 532 Lecture 25

FCH 532 Lecture 25. Chapter 26: Amino acid metabolism Quiz Friday Glucogenic/Ketogenic amino acids (15 min) Quiz Monday April 2:Translation factors Exam 3 on Monday, April 9. Figure 32-45 Translational initiation pathway in E. coli. 50S and 30S associated.

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FCH 532 Lecture 25

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  1. FCH 532 Lecture 25 Chapter 26: Amino acid metabolism Quiz Friday Glucogenic/Ketogenic amino acids (15 min) Quiz Monday April 2:Translation factors Exam 3 on Monday, April 9.

  2. 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

  3. Page 1327

  4. 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

  5. Page 1322

  6. Trp is both glucogenic and ketogenic • Trp is broken down into Ala (pyruvate) and acetoacetate. • First 4 reactions lead to Ala and 3-hydroxyanthranilate. • Reactions 5-9 convert 3-hydroxyanthranilate to a-ketoadipate. • Reactions 10-16 are catalyzed by enzymes of reactions 5 - 11 in Lys degradation to yield acetoacetate.

  7. Page 1007

  8. Page 1007 1. Tryptophan-2,3-dioxygenase, 2. Formamidase, 3. Kynurenine-3-monooxygense, 4. kynureninase (PLP dependent)

  9. Kynureinase, another PLP mechanism • Reaction 4: cleavage of 3-hydroxykynurenine to alanine and 3-hydroxyanthranilate is catalyzed by the PLP dependent enzyme kynureinase. • This facilitates a C-C bond cleavage. (previous reactions catalyzed the C-H and C-C bond cleavage) • Follows the same steps as transamination but does not hydrolyze the tautomerized Schiff base. • Enzyme amino acid acts as a nucleophile tto attack the carbonyl carbon (Cof the tautomerized 3-hydroxykynurenine-PLP Schiff base.

  10. Page 1008

  11. Page 1007 6. Amino carboxymuconate semialdehyde decarboxylase 7. Aminomuconate semialdehyde dehydrogenase 8. Hydratase, 9. Dehydrogense 10-16. Reactions 5-11 in lysine degradation.

  12. -keto acid dehydrogenase • Glutaryl-CoA dehydrogenase • Decarboxylase • Enoyl-CoA hydratase • -hydroxyacyl-CoA dehydrogenase • HMG-CoA synthase • HMG-CoA lyase Page 1006

  13. Phe and Tyr are degraded to fumarate and acetoacetate • The first step in Phe degradation is conversion to Tyr so both amino acids are degraded by the same pathway. • 6 reactions

  14. Phenylanalnine hydroxylase • Aminotransferase • p-hydroxyphenylpyruvate dioxygenase • Homogentisate dioxygenase • Maleylacetoacetate isomerase • Fumarylacetoacetase Page 1009

  15. Phenylalanine hydroxylase has biopterin cofactor • 1st reaction is a hydroxylation reaction by phenylalanine hydroxylase (PAH), a non-heme-iron containing homotetrameric enzyme. • Requires O2, FeII, and biopterin a pterin derivative. • Pterins have a pteridine ring (similar to flavins) • Folate derivatives (THF) also contain pterin rings.

  16. Figure 26-27 The pteridine ring, the nucleus of biopterin and folate. Page 1009

  17. Active BH4 must be regenerated • Active form in PAH is 5,6,7,8-tetrahydrobiopterin (BH4) • Produced from 7,8-dihydrobiopterin via dihydrofolate reductase (NADPH dependent). • 5,6,7,8-tetrahydrobiopterin is hydroxylated to pterin-4a-cabinolamine by phenylalanine hydroxylase. • pterin-4a-cabinolamine is converted to 7,8-dihydrobiopterin (quinoid form) by pterin-4a-carbinoline dehydratase • 7,8-dihydrobiopterin (quinoid form) is reduced by dihydropteridine reductase to regenerate the active cofactor.

  18. Page 1010

  19. NIH shift • A 3H that starts on C4 of Phe’s ring ends up on C3 of Tyr’s ring rather than being lost to solvent. • Mechanism is called the NIH shift • 1st characterized by scientists at NIH

  20. 1 and 2: activation of the enzyme’s BH4 and Fe(II) cofactors to yield pterin-4a-carbinolamine and a reactive oxyferryl [Fe(IV)=O2-] 3: Fe(IV)=O2- reacts with Phe to form an epoxide across the 3,4 bond. 4: epoxide opening to form carbocation at C3 5: migration of hydride from C4 to C3 to form more stable carbocation. 6: ring aromatization to form Tyr

  21. Phe and Tyr are degraded to fumarate and acetoacetate • The first step in Phe degradation is conversion to Tyr so both amino acids are degraded by the same pathway. • 6 reactions • Reaction 1 = 1st NIH shift • Reaction 3 is also an example of NIH shift (26-31)

  22. Phenylanalnine hydroxylase • Aminotransferase • p-hydroxyphenylpyruvate dioxygenase • Homogentisate dioxygenase • Maleylacetoacetate isomerase • Fumarylacetoacetase Page 1009

  23. Amino acid biosynthesis • Essential amino acids - amino acids that can only be synthesized in plants and microorganisms. • Nonessential amino acids - amino acids that can be synthesized in mammals from common intermediates.

  24. Table 26-2Essential and Nonessential Amino Acids in Humans. Page 1030

  25. Nonessential amino acid biosynthesis • Except for Tyr, pathways are simple • Derived from pyruvate, oxaloacetate, -ketoglutarate, and 3-phosphoglycerate. • Tyrosine is misclassified as nonessential since it is derived from the essential amino acid, Phe.

  26. Glutamate biosynthesis • Glu synthesized by Glutamate synthase. • Occurs only in microorganisms, plants, and lower animals. • Converts -ketoglutarate and ammonia from glutamine to glutamate. • Reductive amination requires electrons from either NADPH or ferredoxin (organism dependent). • NADPH-dependent glutamine synthase from Azospirillum brasilenseis the best characterized enzyme. • Heterotetramer (22) with FAD, 2[4Fe-4S] clusters on the  subunit and FMN and [3Fe-4S] cluster on the subunit • NADPH + H+ + glutamine + -ketoglutarate  2 glutamate + NADP+

  27. Figure 26-51 The sequence of reactions catalyzed by glutamate synthase. Electrons are transferred from NADPH to FAD at active site 1 on the  subunit to yield FADH2. Electrons transferred from FADH2 to FMN on site 2 to yield FMNH2. Gln is hydrolyzed to -glutamate and ammonia on site 3 of the  subunit. Ammonia is transferred to site 2 to form -iminoglutarate from -KG -iminoglutarate is reduced by FMNH2 to form glutamate. Page 1031

  28. Figure 26-52 X-Ray structure of the a subunit of A. brasilense glutamate synthase as represented by its Ca backbone. Page 1032

  29. Figure 26-53 The  helix of A. brasilense glutamate synthase. C-terminal domain of glutamate synthase is a 7-turn, right-handed  helix. 43 angstrom long. Structural role for the passage of ammonia. Page 1032

  30. Ala, Asn, Asp, Glu, and Gln are synthesized from pyruvate, oxaloacetate, and -ketoglutarate • Pyruvate is the precursor to Ala • Oxaloacetate is the precursor to Asp • -ketoglutarate is the precursor to Glu • Asn and Gln are synthesized from Asp and Glu by amidation.

  31. Figure 26-54 The syntheses of alanine, aspartate, glutamate, asparagine, and glutamine. Page 1033

  32. Gln and Asn synthetases • Glutamine synthetase catalyzes the formation of glutamine in an ATP dependent manner (ATP to ADP + Pi). • Makes glutamylphosphate intermediate. • NH4+ is the amino group donor. • Asparagine synthetase uses glutamine as the amino donor. • Hydrolyzes ATP to AMP + PPi

  33. Glutamine synthetase is a central control point in nitrogen metabolism • Gln is an amino donor for many biosynthetic products and also a storage compound for excess ammonia. • Mammalian glutamine synthetase is activated by ketoglutarate. • Bacterial glutamine synthetase has more complicated regulation. • 12 identical subunits, 469-aa, D6 symmetry. • Regulated by different effectors and covalent modification.

  34. Figure 26-55a X-Ray structure of S. typhimurium glutamine synthetase. (a) View down the 6-fold axis showing only the six subunits of the upper ring. Active sites shown w/ Mn2+ ions (Mg2+) Adenylation site is indicated in yellow (Tyr) ADP is shown in cyan and phosphinothricin is shown (Glu inhibitor) Page 1034

  35. Figure 26-55b Side view of glutamine synthetase along one of the enzyme’s 2-fold axes showing only the eight nearest subunits. Page 1034

  36. Glutamine synthetase regulation • 9 feedback inhibitors control the activity of bacterial glutamine synthetase • His, Trp, carbamoyl phosphate, glucosamine-6-phosphate, AMP and CTP-pathways leading away from Gln • Ala, Ser, Gly-reflect cell’s N level • Ala, Ser, Gly, are competitive with Glu for the binding site. • AMP and CTP are competitive with the ATP binding site.

  37. Glutamine synthetase regulation • E. coli glutmine synthetase is covalently modified by adenylation of a Tyr. • Increases susceptiblity to feedback inhibition and decreases activity dependent on adenylation. • Adenylation and deadenylation are catalyzed by adenylyltransferase in complex with a tetrameric regulatory protein, PII. • Adensyltransferase deadenylates glutamine synthetase when PII is uridylated. • Adenylates glutamine synthetase when PII lacks UM residues. • PII uridylation depends on the activities of a uridylyltransferase and uridylyl-removing enzyme that hydrolyzes uridylyl groups.

  38. Glutamine synthetase regulation • Uridylyltransferase is activated by -ketoglutarate and ATP. • Uridylyltransferase is inhibited by glutamine and Pi. • Uridylyl-removing enzyme is insensitive to these compounds.

  39. Figure 26-56 The regulation of bacterial glutamine synthetase. Page 1035

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