Rapid Kinetic Analysis of Protein Synthesis - Biophysical Approaches to Translational Control of Gene Expression - page 124

A plausible model which was supported by structural and computational analysis
suggested a shuttle of protons between the attacking nucleophile, the 2¢ OH and 3¢
OH groups of A76; in this model, the ribosome would provide a favorable electro-
static environment that stabilizes the substrates, products, and the TS (Wallin and
Aqvist 2010 ). However, the exact pathway of proton transfer and the number of
protons actually involved remained unclear.
The next important step in unraveling the mechanism of peptide bond formation
was accomplished by chemical synthesis of substrate derivatives and kinetic stud-
ies. The detailed analysis of heavy-atom isotope effects using a set of pept-tRNA
derivatives with substitutions of essentially every atom taking part in catalysis has
demonstrated that the ribosome contributes to chemical catalysis by changing the
rate-limiting TS (Hiller et al. 2011 ; Kingery et al. 2008 ). In solution, the reaction is
rate-limited by the deprotonation of the T ± intermediate at low pH and by the
decomposition of the T − intermediate at high pH (Satterthwait and Jencks 1974 ) . In
contrast, on the ribosome the formation of the tetrahedral intermediate and proton
transfer from the nucleophilic nitrogen both take place during the rate-limiting
step; the breakdown of the tetrahedral intermediate occurs in a separate fast step
(Hiller et al. 2011 ). This result would argue against a fully concerted shuttle where
the formation of the 3¢-leaving group occurs at the same time as a proton from the
a-amino group is received by the 2¢ OH of A76 of pept-tRNA. This is important,
as the 2¢ OH group can receive a proton only if it simultaneously donates a proton
to some other group. However, model studies suggested that the carbonyl oxygen
of the pept-tRNA, rather than the 3¢ OH, accepts the proton from the 2¢ OH (Huang
et al. 2008 ; Rangelov et al. 2006 ). Such a shuttle mechanism may be six-mem-
bered, with two protons simultaneously changing their positions in the TS, or
eight-membered with three protons “in flight” in the TS; the latter TS includes a
water molecule which is found in the right position in the crystal structures of the
50S subunits in the complex with TS analogs (Schmeing et al. 2005a ; Wallin and
Aqvist 2010 ). The analysis of kinetic solvent isotope effects showed that three
protons move in the rate-limiting TS and that the movement is concerted
(Kuhlenkoetter et al. 2011 ). This not only supports the existence of a concerted
proton shuttle, but also favors an eight-membered shuttle with a water molecule
serving as intermediary. The results of the kinetic analysis of pH profiles, muta-
tional and chemical substitutions, kinetic isotope effects, in combination with the
structural and computational work suggest the following mechanism of peptide
bond formation (Fig. 7.1e ). In the rate-limiting TS, the attack of the a -amino group
on the ester carbon results in an eight-membered transition state, in which a proton
from the a-amino group is received by the 2¢ OH group of A76, which at the same
time donates its proton to the carbonyl oxygen through an adjacent water molecule.
Such a scenario would not require a p K a shift of the 2¢ OH group, due to the con-
certed nature of the bond-forming and bond-breaking events. Protonation of the 3¢
OH then would be an independent rapid step. The ribosome catalyzes the reaction
by providing a network of interactions that change the rate-limiting TS state and
lower the entropy of activation.