Journal
BIOCHEMISTRY
Volume 49, Issue 18, Pages 3868-3878Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi901458x
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Funding
- National Institutes of Health (NIH) [GM54839, GM079980]
- Brown-Coxe fellowship
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The ester bond of peptidyl-tRNA undergoes nucleophilic attack in solution and when catalyzed by the ribosome. To characterize the uncatalyzed hydrolysis reaction, a model of peptide release, the transition state structure for hydrolysis of a peptidyl-tRNA mimic was determined. Kinetic isotope effects were measured at several atoms that, potentially undergo a change in bonding in the transition state. Large kinetic isotope effects of carbonyl O-18 and alpha-deuterium substitutions on uncatalyzed hydrolysis indicate the transition state is nearly tetrahedral. Kinetic isotope effects were also measured for aminolysis by hydroxylamine to study a reaction similar to the formation of a peptide bond. In contrast to hydrolysis, the large leaving group O-18 isotope effect indicates the C-O3' bond has undergone significant scission in the transition state. The smaller carbonyl O-18 and alpha-deuterium effects are consistent with a later transition state. The assay developed here can also be used to measure isotope effects for the ribosome-catalyzed reactions. These uncatalyzed reactions serve as a basis for determining what aspects of the transition states are stabilized by the ribosome to achieve a rate enhancement.
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