FCH 532 Lecture 25

Chapter 26: Amino acid metabolismQuiz Friday Glucogenic/Ketogenic amino acids

(15 min)Quiz Monday April 2:Translation factors Exam 3 on Monday, April 9.

Figure 32-45Translational initiation pathway in E. coli.

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• 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.

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RF-1 = UAARF-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

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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.

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

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 (Cof the tautomerized 3-hydroxykynurenine-PLP Schiff base.

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6. Amino carboxymuconate semialdehyde decarboxylase

7. Aminomuconate semialdehyde dehydrogenase

8. Hydratase, 9. Dehydrogense 10-16. Reactions 5-11 in lysine degradation.

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• -keto acid dehydrogenase

• Glutaryl-CoA dehydrogenase

• Decarboxylase

• Enoyl-CoA hydratase

• -hydroxyacyl-CoA dehydrogenase

• HMG-CoA synthase

• HMG-CoA lyase

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

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1. Phenylanalnine hydroxylase2. Aminotransferase3. p-hydroxyphenylpyruvate

dioxygenase4. Homogentisate dioxygenase5. Maleylacetoacetate isomerase6. Fumarylacetoacetase

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.

Figure 26-27The pteridine ring, the

nucleus of biopterin and

folate.

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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.

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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

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

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)

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1. Phenylanalnine hydroxylase2. Aminotransferase3. p-hydroxyphenylpyruvate

dioxygenase4. Homogentisate dioxygenase5. Maleylacetoacetate isomerase6. Fumarylacetoacetase

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.