FCH 532 Lecture 26
Chapter 26: Essential amino acidsQuiz Monday: Translation factorsQuiz Wed: NIH ShiftQuiz Fri: Essential amino acidsExam 3: Next Monday
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.
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.
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 (22) 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+
Figure 26-51The sequence of reactions catalyzed by glutamate synthase.
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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.
Figure 26-52X-Ray structure of the subunit of A. brasilense glutamate synthase as represented by its C
backbone.
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Figure 26-53The helix of A. brasilense glutamate synthase.
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C-terminal domain of glutamate synthase is a 7-turn, right-handed helix.
43 angstrom long.
Structural role for the passage of ammonia.
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 26-54The syntheses of alanine, aspartate, glutamate,
asparagine, and glutamine.
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
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.
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.
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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)
Figure 26-55b Side view of glutamine synthetase along one of the enzyme’s 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-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.
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.
Glutamine synthetase regulation
• Uridylyltransferase is activated by -ketoglutarate and ATP.
• Uridylyltransferase is inhibited by glutamine and P i.• Uridylyl-removing enzyme is insensitive to these
compounds.
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Figure 26-56The regulation of bacterial glutamine synthetase.
Figure 26-57The 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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1. -glutamyl kinase
2. Dehydrogenase
3. Nonenzymatic
4. Pyrroline-5-carboxylate reductase
Proline synthesis
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
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5.glutamyl kinase
6. Acetylglutamate kinase
• N-acetyl--glutamyl phosphate dehydrogense
• N-acetylornithine--aminotransferase
• Acetylornithine deacetylase
• ornithine--aminotransferase
• Urea cycle to arginine
Arginine synthesis
Figure 26-58The conversion of glycolytic intermediate 3-
phosphoglycerate to serine.
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1. Conversion of 3-phosphoglycerate’s 2-OH group to a ketone
2. Transamination of 3-phosphohydroxypyruvate to 3-phosphoserine
3. Hydrolysis of phosphoserine to make Ser.
Serine is the precursor for Gly
• Ser can act in glycine synthesis in two ways:1. Direct conversion of serine to glycine by hydroxymethyl transferase in
reverse (also yields N5, N10-methylene-THF)
2. Condensation of the N5, N10-methylene-THF with CO2 and NH4+ by the glycine
cleavage system
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)
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.
Figure 26-59a Cysteine biosynthesis. (a) The
synthesis of cysteine from serine in plants and
microorganisms.
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Figure 26-59bCysteine biosynthesis. (b) The 8-electron reduction of sulfate to sulfide in E. coli.
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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
Biosynthesis of essential amino acids
• Pathways only present in microorganisms and plants.• Derived from metabolic precursors.• Usually involve more steps than nonessential amino acids.
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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biosynthesis of the “aspartate family” of amino acids: lysine,
methionine, and threonine.
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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.
Figure 26-63The
biosynthesis of phenylalanine, tryptophan, and
tyrosine from chorismate.
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Figure 26-64A ribbon diagram of the bifunctional enzyme tryptophan synthase from S. typhimurium
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Figure 26-65The biosynthesis of
histidine.
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