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FCH 532 Lecture 26

FCH 532 Lecture 26. Chapter 26: Essential amino acids Quiz Monday: Translation factors Quiz Wed: NIH Shift Quiz Fri: Essential amino acids Exam 3: Next Monday. Amino acid biosynthesis. Essential amino acids - amino acids that can only be synthesized in plants and microorganisms.

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FCH 532 Lecture 26

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  1. FCH 532 Lecture 26 Chapter 26: Essential amino acids Quiz Monday: Translation factors Quiz Wed: NIH Shift Quiz Fri: Essential amino acids Exam 3: Next Monday

  2. 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.

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

  4. 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.

  5. 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+

  6. 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

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

  8. 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

  9. 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.

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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

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

  19. Figure 26-57 The biosynthesis of the “glutamate family” of amino acids: arginine, ornithine, and proline. Page 1036

  20. Conversion of Glu to Pro • Involves reduction of the -carboxyl group to an aldehyde followed for the formation of an internal Schiff base. This is reduced to make Pro.

  21. Proline synthesis -glutamyl kinase Dehydrogenase Nonenzymatic Pyrroline-5-carboxylate reductase Page 1036

  22. Glutamate is the precursor for Proline, Ornithine, and Arginine • E. coli pathway from Gln to ornithine and Arg involves ATP-driven reduction of the glutamate gamma carboxyl group to an aldehyde (N-acetylglutamate-5-semialdehyde). • Spontaneous cyclization is prevented by acetylation of amino group by N-acetylglutamate synthase. • N-acetylglutamate-5-semialdehyde is converted to amine by transamination. • Hydrolysis of protecting group yields ornithine which can be converted to arginine. • In humans it is direct from glutamate-5-semialdehyde to ornithine by ornithine--aminotransferase

  23. Arginine synthesis glutamyl kinase 6. Acetylglutamate kinase N-acetyl--glutamyl phosphate dehydrogense N-acetylornithine--aminotransferase Acetylornithine deacetylase ornithine--aminotransferase Urea cycle to arginine Page 1036

  24. Figure 26-58 The conversion of glycolytic intermediate 3-phosphoglycerate to serine. Conversion of 3-phosphoglycerate’s 2-OH group to a ketone Transamination of 3-phosphohydroxypyruvate to 3-phosphoserine Hydrolysis of phosphoserine to make Ser. Page 1037

  25. Serine is the precursor for Gly • Ser can act in glycine synthesis in two ways: • Direct conversion of serine to glycine by hydroxymethyl transferase in reverse (also yields N5, N10-methylene-THF) • Condensation of the N5, N10-methylene-THF with CO2 and NH4+ by the glycine cleavage system

  26. Cys derived from Ser • In animals, Cys is derived from Ser and homocysteine (breakdown product of Met). • The -SH group is derived from Met, so Cys can be considered essential.

  27. Methionine adenosyltransferase Methyltransferase Adenosylhomocysteinase Methionine synthase (B12) Cystathionine -synthase (PLP) Cystathionine -synthase (PLP) -ketoacid dehydrogenase Propionyl-CoA carboxylase (biotin) Methylmalonyl-CoA racemase Methylmalonyl-CoA mutase Glycine cleavage system or serine hydroxymethyltransferase N5,N10-methylene-tetrahydrofolate reductase (coenzyme B12 and FAD) Page 1002

  28. Cys derived from Ser • In plants and microorganisms, Cys is synthesized from Ser in two step reaction. • Reaction 1: activation of Ser -OH group by converting to O-acetylserine. • Reaction 2: displacement of the acetate by sulfide. • Sulfide is derived fro man 8-electron reduction reaction.

  29. Figure 26-59a Cysteine biosynthesis. (a) The synthesis of cysteine from serine in plants and microorganisms. Page 1038

  30. Figure 26-59b Cysteine biosynthesis. (b) The 8-electron reduction of sulfate to sulfide in E. coli. Sulfate activation by ATP sulfuylase and adeosine-5’-phosphosulfate (APS) kinase Sulfate reduced to sulfite by 3’-phosphoadenosine-5’-phosphosulfate (PAPS) reductase Sulfite to sulfide by sulfite reductase Page 1038

  31. Biosynthesis of essential amino acids • Pathways only present in microorganisms and plants. • Derived from metabolic precursors. • Usually involve more steps than nonessential amino acids.

  32. Biosynthesis of Lys, Met, Thr • First reaction is catalyzed by aspartokinase which converts aspartate to apartyl--phosphate. • Each pathway is independently controlled.

  33. Figure 26-60 The biosynthesis of the “aspartate family” of amino acids: lysine, methionine, and threonine. Page 1039

  34. Figure 26-61 The biosynthesis of the “pyruvate family” of amino acids: isoleucine, leucine, and valine. Page 1040

  35. Figure 26-62 The biosynthesis of chorismate, the aromatic amino acid precursor. Page 1042

  36. Figure 26-63 The biosynthesis of phenylalanine, tryptophan, and tyrosine from chorismate. Page 1043

  37. Figure 26-64 A ribbon diagram of the bifunctional enzyme tryptophan synthase from S. typhimurium Page 1044

  38. Figure 26-65 The biosynthesis of histidine. Page 1045

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