FCH 532 Lecture 26

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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

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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.

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

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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 brasilense is the best characterized
  enzyme.
• Heterotetramer (?2?2) with FAD, 24Fe-4S
  clusters on the ? subunit and FMN and 3Fe-4S
  cluster on the ??subunit
• NADPH H glutamine ?-ketoglutarate ? 2
  glutamate NADP

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Figure 26-51 The sequence of reactions catalyzed
by glutamate synthase.

1. Electrons are transferred from NADPH to FAD at
   active site 1 on the ? subunit to yield FADH2.
2. Electrons transferred from FADH2 to FMN on site 2
   to yield FMNH2.
3. Gln is hydrolyzed to ?-glutamate and ammonia on
   site 3 of the ? subunit.
4. Ammonia is transferred to site 2 to form
   ?-iminoglutarate from ?-KG
5. ?-iminoglutarate is reduced by FMNH2 to form
   glutamate.

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

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

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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.

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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)
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Figure 26-55b Side view of glutamine synthetase
along one of the enzymes 2-fold axes showing
only the eight nearest subunits.
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Glutamine synthetase regulation

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

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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.

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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.

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Figure 26-56 The regulation of bacterial
glutamine synthetase.
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Figure 26-57 The biosynthesis of the glutamate
family of amino acids arginine, ornithine, and
proline.
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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.

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Proline synthesis

1. ?-glutamyl kinase
2. Dehydrogenase
3. Nonenzymatic
4. Pyrroline-5-carboxylate reductase

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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-semialdehy
  de to ornithine by ornithine-?-aminotransferase

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Arginine synthesis

• ?????glutamyl kinase
• 6. Acetylglutamate kinase
• N-acetyl-?-glutamyl phosphate dehydrogense
• N-acetylornithine-?-aminotransferase
• Acetylornithine deacetylase
• ornithine-?-aminotransferase
• Urea cycle to arginine

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Figure 26-58 The conversion of glycolytic
intermediate 3-phosphoglycerate to serine.

1. Conversion of 3-phosphoglycerates 2-OH group to
   a ketone
2. Transamination of 3-phosphohydroxypyruvate to
   3-phosphoserine
3. Hydrolysis of phosphoserine to make Ser.

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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

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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.

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1. Methionine adenosyltransferase
2. Methyltransferase
3. Adenosylhomocysteinase
4. Methionine synthase (B12)
5. Cystathionine ?-synthase (PLP)
6. Cystathionine ?-synthase (PLP)
7. ?-ketoacid dehydrogenase
8. Propionyl-CoA carboxylase (biotin)
9. Methylmalonyl-CoA racemase
10. Methylmalonyl-CoA mutase
11. Glycine cleavage system or serine
    hydroxymethyltransferase
12. N5,N10-methylene-tetrahydrofolate reductase
    (coenzyme B12 and FAD)

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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.

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Figure 26-59a Cysteine biosynthesis. (a) The
synthesis of cysteine from serine in plants and
microorganisms.
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Figure 26-59b Cysteine biosynthesis. (b) The
8-electron reduction of sulfate to sulfide in E.
coli.

1. Sulfate activation by ATP sulfuylase and
   adeosine-5-phosphosulfate (APS) kinase
2. Sulfate reduced to sulfite by 3-phosphoadenosine-
   5-phosphosulfate (PAPS) reductase
3. Sulfite to sulfide by sulfite reductase

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Biosynthesis of essential amino acids

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

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Biosynthesis of Lys, Met, Thr

• First reaction is catalyzed by aspartokinase
  which converts aspartate to apartyl-?-phosphate.
• Each pathway is independently controlled.

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Figure 26-60 The biosynthesis of the aspartate
family of amino acids lysine, methionine, and
threonine.
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Figure 26-61 The biosynthesis of the pyruvate
family of amino acids isoleucine, leucine, and
valine.
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Figure 26-62 The biosynthesis of chorismate, the
aromatic amino acid precursor.
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Figure 26-63 The biosynthesis of phenylalanine,
tryptophan, and tyrosine from chorismate.
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Figure 26-64 A ribbon diagram of the bifunctional
enzyme tryptophan synthase from S. typhimurium
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Figure 26-65 The biosynthesis of histidine.
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