FCH 532 Lecture 21
Chapter 32: Translation
Quiz today (Wed.) on amino acids
Quiz on Friday TCA cycle
Quiz on Monday Transamination mechanism.
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Figure 32-43 Some translational initiation (ShineDalgarno) sequences recognized by E. coli ribosomes.
Shine-Dalgarno sequences typically start 10-15 nt upstream
of the initiation codon.
Are only found in prokaryotes.
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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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Defining tRNA Binding Sites in
Different functional States
GENERATE RIBOSOMES IN THE FOLLOWING STATES:
A (Aminoacyl): EF-Tu.GTP dependent; mRNA dependent;
occupied P Site
P (Peptidyl): Reactive with Puromycin (Pm)
E (Exit): Deacylated tRNA
MONITOR BY CHEMICAL FOOTPRINTING:
30S A site protections:
50S P site protections
(also X-linkers, EDTA-FeII)
Looking at footprint pre and post peptide bond, translocation
The data didn't fit into a simple 2 site model
HYBRID STATES HAD TO BE INVOKED
tRNA movement occurs independently on 2
subunits via 6 hybrid states.
1. A/T --> 2. A/A --> 3. A/P --> 4. P/P --> 5. P/E --> 6. E
In this model the tRNA would "ratchet" its way through the
ribosome undergoing 50° rotations along its longitudinal
axis from A to P.
This model has received support from EM and X-ray studies.
cryo-EM
Aminoacyl-tRNA
EF-Tu-GTP
EF-Ts
GTP
EF-Tu-EF-Ts
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EF-Ts
GDP
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
Translation
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Shine-Dalgarno sequence
Initiation
Elongation
Release
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Figure 32-61 Ribosome-catalyzed hydrolysis of
peptidyl–tRNA to form a polypeptide and free tRNA.
Urea Cycle
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Excess nitrogen is excreted after the metabolic breakdown of amino acids in one of
three forms:
Aquatic animals are ammonotelic (release NH3 directly).
If water is less plentiful, NH3 is converted to less toxic products, urea and uric acid.
Terrestrial vertebrates are ureotelic (excrete urea)
Birds and reptiles are uricotelic (excrete uric acid)
Urea is made by enzymes urea cycle in the liver.
The overall reaction is:
NH3+
NH3 + HCO3- + -OOC-CH2-CH-COOAsp
O
3ATP
2ADP + 2Pi + AMP + PPi
NH2-C-NH2 + -OOC-CH=CH-COOUrea
Fumarate
Urea Cycle
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•
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2 urea nitrogen atoms come from ammonia and
aspartate.
Carbon atom comes from bicarbonate.
5 enzymatic reactions used, 2 in the mitochondria and 3
in the cytosol.
NH3+
NH3 + HCO3- + -OOC-CH2-CH-COOAsp
O
3ATP
2ADP + 2Pi + AMP + PPi
NH2-C-NH2 + -OOC-CH=CH-COOUrea
Fumarate
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Carbamoyl phosphate synthetase
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Carbamoyl phosphate synthetase (CPS) catalyzes the
condensation and activation NH3 and HCO3- to form carbomyl
phosphate (first nitrogen containing substrate).
Uses 2 ATPs.
O
2ATP + NH3 + HCO3-  NH2-C-OPO3- + 2ADP + 2Pi
Carbamoyl phosphate
•
1.
2.
Eukaryotes have 2 types of CPS enzymes
Mitochondrial CPSI uses NH3 as its nitrogen donor and participates in urea
biosynthesis.
Cytosolic CPSII uses glutamine as its nitrogen donor and is involved in
pyrimidine biosynthesis.
Figure 26-8 The
mechanism of action of
CPS I.
•
1.
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CPSI reaction has 3 steps
Activation of HCO3- by ATP to form
carboxyphosphate and ADP.
2.
Nucelophilic attack of NH3 on
carboxyphosphate, displacing the
phsophate to form carbamate and
Pi.
3.
Phosphorylation of carbamate by the
second ATP to form carbamoyl
phosphate and ADP
The reaction is irreversible.
Allosterically activated by Nacetylglutamate.
Figure 26-9 X-Ray structure of
E. coli carbamoyl phosphate
synthetase (CPS).
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E. coli has only one CPS
(homology to CPS I and CPS II)
Heterodimer (inactive).
Allosterically activated by ornithine
(heterotetramer of (4).
Small subunit hydrolyzes Gln and
delivers NH3 to large subunit.
Channels intermediate of two
reactions from one active site to the
other.
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Ornithine transcarbomylase
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Transfers the carbomoyl group of carbomyl phosphate to ornithine
to make citrulline
Reaction occurs in mitochondrion.
Ornithine produced in the cytosol enters via a specific transport
system.
Citrulline is exported from the mitochondria.
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Arginocuccinate Synthetase
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2nd N in urea is incorporated in the 3rd reaction of the urea cycle.
Condensation reaction with citrulline’s ureido group with an Asp
amino group catalyzed by arginosuccinate synthetase.
Ureido oxygen is activated as a leaving group through the
formation of a citrulyl-AMP intermediate.
This is displaced by the Asp amino group to form
arginosuccinate.
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Figure 26-10 The mechanism of action of
argininosuccinate synthetase.
Arigininosuccinase and Arginase
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Argininosuccinse catalyzes the elimination of Arg from
the the Asp carbon skeleton to form fumurate.
Arginine is the immediate precursor to urea.
Fumurate is converted by fumarase and malate
dehydrogenase to to form OAA for gluconeogenesis.
Arginase catalyzes the fifth and final reaction of the
urea cycle.
Arginine is hydrolyzed to form urea and regenerate
ornithine.
Ornithine is returned to the mitochondria.
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1. Carbamoyl
phosphate
synthetase (CPS)
2. Ornithine
transcarbamoylase
3. Argininosuccinate
synthetase
4. Arginosuccinase
5. Arginase
Regulation of the urea cycle
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Carbamoyl phosphate synthetase I is allosterically
activated by N-acetylglutamate.
N-acetylglutamate is synthesized from glutamate and acetylCoA by N-acetylglutamate synthase, it is hydrolyzed by a
specific hydrolase.
Rate of urea production is dependent on [N-acetylglutamate].
When aa breakdown rates increase, excess nitrogen must be
excreted. This results in increase in Glu through
transamination reactions.
Excess Glu causes an increase in N-acetylglutamate which
stimulates CPS I causing increases in urea cycle.
Metabolic breakdown of amino
acids
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Degradation of amino acids converts the to TCA cycle
intermediates or precursors to be metabolized to CO2, H2O,
or for use in gluconeogenesis.
Aminoacids are glucogenic, ketogenic or both.
Glucogenic amino acids-carbon skeletons are broken down
to pyruvate, -ketoglutarate, succinyl-CoA, fumarate, or
oxaloacetate (glucose precursors).
Ketogenic amino acids, are broken down to acetyl-CoA or
acetoacetate and therefore can be converted to fatty acids or
ketone bodies.