Rapid Kinetic Analysis of Protein Synthesis - Biophysical Approaches to Translational Control of Gene Expression - page 121

7.5.1 Peptide Bond Formation

On the ribosome, peptide bond formation takes place between two substrates,
pept-tRNA in the P site and aa-tRNA in the A site (Fig. 7.1a ). The reaction proceeds
through the nucleophilic attack of the a-amino group of aa-tRNA on the carbonyl
carbon of the peptidyl-tRNA (Fig. 7.1b ); from a chemical point of view, the reaction
is the aminolysis of an ester. Based on the classical studies with model substrates in
solution, the reaction is expected to proceed through two intermediates (Fig. 7.1c ).
First, a zwitterionic tetrahedral intermediate is formed (T ± ). Deprotonation of the
positively charged amino nitrogen forms the second intermediate (T − ). In the final
step, T − decomposes to form the reaction products (Satterthwait and Jencks 1974 ) ;
for peptide bond formation these are deacylated tRNA in the P site of the ribosome
and a new pept-tRNA, longer by one amino acid, in the A site.
The ribosome could contribute to catalysis in a number of ways, e.g., by stabiliz-
ing charges developing in the transition state (TS) or by acid-base catalysis involv-
ing ionizing groups of the ribosome at the active center, analogous to protein
enzymes that employ amino acid side chains to shuffle protons in the reactions they
catalyze. If the latter were true for the ribosome, the rate of catalysis should depend
on pH in a way that reflects the p K a values of the ionizing group(s) taking part in
catalysis. Furthermore, the pH/rate profiles should be sensitive to substitution (muta-
tions) of the presumed catalytic residues in the ribosome's active site. As the cata-
lytic center of the ribosome does not contain proteins, the large repertoire of
chemically active groups of amino acids that can act as efficient acid/base catalysts
is not available. Instead, rRNA has a very limited choice of groups that would be
useful for catalysis at physiological pH, as their p K a values are far from neutrality.
This renders rRNA bases inefficient as acid/base catalysts, unless their p K a values
were shifted towards neutrality in the environment of the ribosome. These consider-
ations provide testable predictions which can be addressed by kinetic studies fol-
lowing the rate-pH-dependence of the peptidyl transfer reaction. These experiments
illustrate the use of the quench-flow technique for addressing a reaction mechanism.
The setup for these experiments was quite simple: Pept-tRNA and aa-tRNA were
allowed to react for a defined time at a given pH, the reaction was stopped, and the
concentrations of reactants and products were analyzed for each time point. Using
radioactively labeled substrates, e.g., [ 14 C] and [ 3 H]-labeled amino acids, made the
analysis straightforward and accurate (Fig. 7.1d ) (Katunin et al. 2002a ; Wohlgemuth
et al. 2008 ) .
One important issue for mechanistic studies of peptide bond formation has been
the optimum choice of the reaction substrates. Before peptide bond formation takes
place, both substrates, the pept-tRNA and aa-tRNA, should be correctly bound at
active site. The simplest biologically relevant P-site substrate is fMet-tRNA fMet ,
which is placed into the P site with the help of the initiation factors. More complex
setups allow the formation of di-, tri-, or oligopeptides in the P site using reconsti-
tuted translation mixtures containing the desired set of aa-tRNAs (Katunin et al.
2002a ; Wohlgemuth et al. 2008 ) (Fig. 7.1d ). Using the natural A-site substrate is