FCH 532 Lecture 25
Chapter 26: Amino acid metabolism
Quiz Friday Glucogenic/Ketogenic amino acids
(15 min)
Quiz Monday April 2:Translation factors
Exam 3 on Monday, April 9.
Page 1323
Figure 32-45 Translational
initiation pathway in E. coli.
• 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 = UAA
RF-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).
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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 3hydroxyanthranilate.
Reactions 5-9 convert 3-hydroxyanthranilate to aketoadipate.
Reactions 10-16 are catalyzed by enzymes of
reactions 5 - 11 in Lys degradation to yield
acetoacetate.
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Page 1007
1. Tryptophan-2,3-dioxygenase, 2. Formamidase, 3.
Kynurenine-3-monooxygense, 4. kynureninase (PLP
dependent)
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•
•
•
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 (Cof the tautomerized 3hydroxykynurenine-PLP Schiff base.
Page
1008
Page 1007
6. Amino carboxymuconate semialdehyde decarboxylase
7. Aminomuconate semialdehyde dehydrogenase
8. Hydratase, 9. Dehydrogense 10-16. Reactions 5-11 in
lysine degradation.
-keto acid
dehydrogenase
•
Glutaryl-CoA
dehydrogenase
•
Decarboxylase
•
Enoyl-CoA
hydratase
•
-hydroxyacyl-CoA
dehydrogenase
•
HMG-CoA
synthase
•
Page 1006
•
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
1.
2.
3.
Page 1009
4.
5.
6.
Phenylanalnine hydroxylase
Aminotransferase
p-hydroxyphenylpyruvate
dioxygenase
Homogentisate dioxygenase
Maleylacetoacetate isomerase
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.
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Figure 26-27 The
pteridine ring, the
nucleus of
biopterin and
folate.
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-4acabinolamine by phenylalanine hydroxylase.
pterin-4a-cabinolamine is converted to 7,8dihydrobiopterin (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-4acarbinolamine 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)
1.
2.
3.
Page 1009
4.
5.
6.
Phenylanalnine hydroxylase
Aminotransferase
p-hydroxyphenylpyruvate
dioxygenase
Homogentisate dioxygenase
Maleylacetoacetate isomerase
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.
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Table 26-2
Essential and Nonessential Amino Acids in
Humans.
Nonessential amino acid
biosynthesis
•
•
•
Except for Tyr, pathways are simple
Derived from pyruvate, oxaloacetate, -ketoglutarate, and 3phosphoglycerate.
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 (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+
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.
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
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1.
5.
-iminoglutarate is
reduced by FMNH2 to
form glutamate.
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Figure 26-52 X-Ray structure of the  subunit of A.
brasilense glutamate synthase as represented by its C
backbone.
Figure 26-53 The  helix of A. brasilense glutamate
synthase.
C-terminal domain of
glutamate synthase is a 7turn, right-handed  helix.
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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 26-54 The
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.
Active sites shown w/
Mn2+ ions (Mg2+)
Adenylation site is
indicated in yellow
(Tyr)
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ADP is shown in
cyan and
phosphinothricin is
shown (Glu inhibitor)
Page 1034
Figure 26-55b
Side view of glutamine
synthetase along one of the enzyme’s 2-fold axes
showing only the eight nearest subunits.
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 Pi.
Uridylyl-removing enzyme is insensitive to these
compounds.
Page 1035
Figure 26-56 The
regulation of bacterial
glutamine synthetase.