title: Transition states of uncatalyzed hydrolysis and aminolysis reactions of a ribosomal P-site substrate determined by kinetic isotope effects.
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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Hiller
authors.forename: Minghong
authors.initials: M
authors.lastname: Zhong
authors.forename: Vipender
authors.initials: V
authors.lastname: Singh
authors.forename: Scott A
authors.initials: SA
authors.lastname: Strobel
mesh_terms: D006868:Hydrolysis; D019811:Hydroxylamine / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D012346:RNA, Transfer, Amino Acyl; C019037:tRNA, peptidyl-; D019811:Hydroxylamine