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I. Structure and Mechanism:
Protein Synthesis
“Mechanism of the peptidyl-transfer
reaction of the prokaryotic ribosome”
By: Trang Bui
Overview
• Introduction-Ribosome
• Structure of Peptidyl Transferase Center
• Mechanism of Peptide Bond Formation
• Acid-base catalysis (enthalpic)
• Proper substrate positioning (entropic)
• Future Prospects
Ribosome
• Prokaryote
– 50S subunit:
• 23S rRNA and 5S rRNA
• 31 proteins
– 30S subunit:
• 26S rRNA
• 21 proteins
• Eukaryote
– 60S subunit:
• 28S rRNA, 5.8S rRNA, &
5S rRNA
• 49 proteins
– 40S subunit:
• 18S rRNA
• 33 proteins
The acceptor ends of A-site and P-site tRNAs are located in the
50S subunit facing the 30S subunit
Review: peptide bond formation
Peptidyl-transferase
• Peptidyl transferase (PT) center
is the site for peptide bond
formation
• Located on 50S
• Composed of RNA only and no
protein is found within 15 Ǻ
What can you conclude from this?
Ribosome Ribozyme
L27 protein might be involved
- deletion of 3 a.a. leads to
impaired activity
- only exits in some organisms
- suggesting that L27 facilitates
the proper placement of the tRNA
at the PTC
Structure of the PT center
•Conserved & are
located at the core of
the PT center
•tRNA analog (A site)
•tRNA analog (P site)
•23S rRNA bases
Knowing that ribosome is a ribozyme,
what mechanism(s) do you think
might be used to form peptide bonds
between 20 different a.a.?
Chemistry of peptide-bond formation
1: Deprotonation of the amino group
2: Nucleophilic attack and formation of the zwitterionic tetrahedral intermediate
3: Deprotonation and formation of the negatively charged intermediate
4: Product formation and protonation of the leaving oxygen
Mechanism of Peptide Bond
Formation
1. Contribution of general acid-base catalysis
2. Contribution of substrate positioning
Acid-base Catalysis
• In general, this means
either proton transfer or
abstraction
• Transition state: chemicals
bonds are in the process of
being made and broken
• Unstable + and – charges
are being developed
• Stabilizing charges
catalyzes the reaction by
lowering the energy of the
transition state
Acid-base Catalysis
• Facilitates the activation of
weak nucleophiles
• Stabilization of poor leaving
groups
Role of active site residue A2451
as general acid-base catalyst
• Crystallized ribosomes with
a transition analog CCdApPuro
• N3 A2451 is in close
contact (3 Ǻ) with a
transition state analog of
peptide bond formation,
CCdA-pPuro
• A2451U mutation led to
significantly reduced activity
• Thought to be involved in
acid-base catalysis
N3 of A2451
• X-ray structures of the
ribosome with a P-site tRNA
A76 2’OH instead of 2’deoxyadenosine
•
Within hydrogen bond
distance of the a-amino
group only in the prereaction state
• Intermediate’s oxyanion
points away from the
A2451-N3
WRONG
A site
P site
23S rRNA bases
Experimental Procedure
Acid-base Catalysis
A site
• Aminoacyl-tRNA binds to the A site in the range of 10 s-1
• The intrinsic rate of peptide-bond formation was estimated to
be > 300 s-1
• Thus, the mechanism of peptide-bond formation cannot be
studied with the native aa-tRNA under current experimental
capabilities.
• Adio et al. (2006) examined the contribution of acid-base
catalysis to peptide bond formation using ribosomes from
E.coli with native aa-tRNA
Experimental Procedure
• The a-amino group of aa-tRNA has a pKa of 8.
• Assuming that an ionization group (pKa = 7) on the ribosome
is involved in the reaction, the rate of the peptide bond
formation should be pH dependent.
• Since the accommodation step is pH independent, the
reaction rate may become lower than the accommodation rate
at a certain pH.
• Measured rates of aa-tRNA accommodation and peptide bond
formation in the pH range between 6 and 9.
• pH<6 = EF-Tu precipitation
• pH>9 = tRNA cleavage
A-site Accommodation
Curve 1: Phe-tRNA (QSY)
Curve 2: Phe-tRNA
• Measuring the rate of A-site accommodation using FRET
– fMet-tRNA was labeled with a fluorescence donor, fluorescein
– Phe-tRNA was labeled with a fluorescence quencher, QSY35
– Time course of accommodation was measured using the
stopped-flow method
• Conclusion: the rates of accommodation were identical at pH
values of 6,7, and 8
Peptide Bond Formation
• Time course of accommodation was measured using the
quench flow method
– f[3H]Met-tRNA
– [14C]Phe-tRNA
• Conclusion: The rate of peptide bond formation was independent of
pH and indistinguishable from the rate of accommodation
Conclusion from pH reactions
• This can be explained in 2 ways:
1) The ionization group has no affect on the rate of the PT
reaction
2) The chemical step was so fast that the contribution from
the protonation step was insignificant
If you can’t study the chemistry step using native aa-tRNA,
what can you do?
Uncoupling the Chemistry Step from
the Accommodation Step
Phelac-tRNA
• -OH is the nucleophile instead of -NH2
• Does not change the catalytic
mechanism
• Formation of ester bond  rate
limiting step
• Measuring the reaction rate at pH 6-9
reveals that the reaction rate is
independent of pH
• This indicates that acid-base catalysis
is not used to a great extent
Peptide Bond Formation
1. Contribution of general acid-base catalysis
2. Contribution of substrate positioning
2’OH of A76
• CCdA-p-Puro inhibits
peptidyl transferase activity
• Substituting 2’-OH of A76
by either 2’-deoxy or 2’fluoro reduce the activity
~10^6 fold
• 2’-OH receives a proton
from the a-amino group
while simultaneously
protonates the leaving
3’OH—proton shuttle
• 2’-OH of A76 orients the
nucleophile
A site tRNA substrate
P site tRNA substrate
Ribosome residues
HOH* might be used for a
proton shuttle
Other Groups Involvement??
2’-OH of A2451
-
Substitution of 2’OH of
A2451 by hydrogen impairs
peptidyl-transferase activity
-
Interacts directly with the
2’OH group of the P-site
tRNA
A site tRNA susbtrates
P site tRNA substrates
Ribosome residues
• The ribosome brings ~10^7 fold enhancement in the rate of
PT reaction compared with the second-order reaction in
solution
• Entropy of activation is lowered
• Enthalpy of activation is the same for both reactions
• In acid-base and covalent catalysis, enzymes act by lowering
the activation enthalpy
Mechanism of Peptide-bond Formation
• Conclusion: entropic catalysis is the major catalytic mechanism
of peptide bond formation
 Intra-reactant proton shuttling via the 2’OH of A76 of
the P-site tRNA
Future Prospects
• Obtain structural and mechanistic information for eukaryotic
ribosomes
• Examine the second important function of the PT center:
termination of protein synthesis