Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511, USA.
Biochemistry. 2010 May 11;49(18):3868-78. doi: 10.1021/bi901458x.
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.
肽酰-tRNA 的酯键在溶液中发生亲核进攻，在核糖体催化下也是如此。为了研究无催化水解反应，即肽释放的过渡态结构，确定了肽酰-tRNA 类似物水解的过渡态结构。在可能在过渡态中发生键合变化的几个原子处测量了动力学同位素效应。羰基 (18)O 和 α-氘取代物对无催化水解的大动力学同位素效应表明过渡态几乎是四面体形的。还通过羟胺进行了氨解反应的动力学同位素效应测量，以研究类似于肽键形成的反应。与水解相反，大的离去基团 (18)O 同位素效应表明在过渡态中 C-O3' 键已发生显著断裂。较小的羰基 (18)O 和 α-氘效应与较晚的过渡态一致。这里开发的测定法也可用于测量核糖体催化反应的同位素效应。这些无催化反应为确定核糖体稳定过渡态的哪些方面以实现速率增强提供了基础。
