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 32-43Some translational initiation (Shine-Dalgarno) sequences recognized by E. coli ribosomes.

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Shine-Dalgarno sequences typically start 10-15 nt upstream of the initiation codon.

Are only found in prokaryotes.

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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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 modelHYBRID 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

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EF-Ts

EF-Tu-EF-Ts

GDP

GTP

EF-Ts

EF-Tu-GTP

Aminoacyl-tRNA

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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

Translation• Shine-Dalgarno sequence• Initiation• Elongation• Release

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Figure 32-61Ribosome-catalyzed hydrolysis of peptidyl–tRNA to form a polypeptide and free tRNA.

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Urea Cycle• 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 + HCO3- + -OOC-CH2-CH-COO-

NH2-C-NH2 + -OOC-CH=CH-COO-

NH3+

3ATP

2ADP + 2Pi + AMP + PPi

Asp

FumarateUrea

O

Urea Cycle• 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 + HCO3- + -OOC-CH2-CH-COO-

NH2-C-NH2 + -OOC-CH=CH-COO-

NH3+

3ATP

2ADP + 2Pi + AMP + PPi

Asp

FumarateUrea

O

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Carbamoyl phosphate synthetase• Carbamoyl phosphate synthetase (CPS) catalyzes the

condensation and activation NH3 and HCO3- to form carbomyl

phosphate (first nitrogen containing substrate).• Uses 2 ATPs.

2ATP + NH3 + HCO3- NH2-C-OPO3

- + 2ADP + 2Pi

Carbamoyl phosphate

O

• Eukaryotes have 2 types of CPS enzymes1. Mitochondrial CPSI uses NH3 as its nitrogen donor and participates in urea

biosynthesis.2. Cytosolic CPSII uses glutamine as its nitrogen donor and is involved in

pyrimidine biosynthesis.

Figure 26-8 The mechanism of action of

CPS I.

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• CPSI reaction has 3 steps1. 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 N-

acetylglutamate.

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• 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• 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.

Figure 26-10The mechanism of action of argininosuccinate synthetase.

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Arigininosuccinase and Arginase

• 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. Arginosuccinase5. Arginase

Regulation of the urea cycle

• Carbamoyl phosphate synthetase I is allosterically activated by N-acetylglutamate.

• N-acetylglutamate is synthesized from glutamate and acetyl-CoA 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

• 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.