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Biochemistry. 2010 May 11;49(18):3868-78. doi: 10.1021/bi901458x.

Transition states of uncatalyzed hydrolysis and aminolysis reactions of a ribosomal P-site substrate determined by kinetic isotope effects.

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  • 1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511, USA.

Abstract

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 (18)O 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 (18)O isotope effect indicates the C-O3' bond has undergone significant scission in the transition state. The smaller carbonyl (18)O 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.

PMID:
20359191
[PubMed - indexed for MEDLINE]
PMCID:
PMC2864349
Free PMC Article
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