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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