Rapid Kinetic Analysis of Protein Synthesis - Biophysical Approaches to Translational Control of Gene Expression - page 120

7.4.3 Design and Evaluation of Experiments

The design of a transient kinetics experiment for a multicomponent reaction can be
quite complex. Generally, the experiment should be planned in such a way that
pseudo-first-order reaction conditions are fulfilled, which greatly simplifies the
calculation of rate constants from observed rates. This is accomplished by taking
limiting concentrations of the labeled component and adding all other reaction
components in large excess, such that the change in concentration of unlabeled com-
ponents during the reaction is negligible. Apparent rate constants of the reaction
( k app or k obs ) are measured at constant concentration of the labeled component and
increasing excess concentrations of its ligands, and the rate constants of the reaction
under study are derived from the analysis of the concentration dependence of the
apparent rate constants. In recent years, the complexities of data analysis have been
overcome by advances in computational methods to analyze reaction time courses
by numerical integration using Matlab or specialized programs (Scientist, Berkeley
Madonna, Dynafit, Kinsim, and numerous others). This allows using equimolar
concentrations of reagents and determining rate constants by global fitting; however,
measuring time courses at a number of different concentrations of reagents is even
more important in that case. Data from different approaches should be combined for
global analysis of a complex reaction.

7.5 Insights into the Catalytic Center of the Ribosome

The catalytic center of the ribosome has to perform two functions: the catalysis of
peptide bond formation during elongation and the hydrolysis of the ester bond in
pept-tRNA in the termination phase of protein synthesis. In addition, ribosomes are
able to catalyze a number of other, unnatural chemical reactions, e.g., thioester,
thioamide, or phosphinoamide formation (Rodnina et al. 2006 ) . Thus, the question
arises of how the same active site can be utilized to catalyze such a variety of differ-
ent chemical reactions. Are the catalytic mechanisms the same or different for pep-
tide bond formation and peptidyl-tRNA hydrolysis? What is the contribution of the
ribosome to catalysis?
The recent history of studies on the catalytic mechanisms of the ribosome goes
back to 2000 when the first crystal structures of the 50S ribosomal subunit from
Haloarcula marismortui were obtained in the complex with a transition state analog
(Nissen et al. 2000 ). The structural models have established that the catalytic center
consists of rRNA and there are no proteins in the vicinity that could donate func-
tional groups to take part in the reaction. The crystal structures provided an excel-
lent framework to formulate and test hypotheses on the mechanism of catalysis.
Because the reactions in vivo must be rapid to account for the measured speed of
protein synthesis (about 10 s −1 ), using rapid kinetic methods is crucial to address
these questions. In the following, we summarize those approaches and the outcome
of those studies in conjunction to further structural, genetic, and chemical work.