Kinetic isotope effect analysis of the ribosomal peptidyl transferase reaction

被引:37
作者
Seila, AC [1 ]
Okuda, K [1 ]
Núñez, S [1 ]
Seila, AF [1 ]
Strobel, SA [1 ]
机构
[1] Yale Univ, Dept Mol Biophys & Biochem, New Haven, CT 06520 USA
关键词
D O I
10.1021/bi047742f
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The ribosome is the macromolecular machine responsible for protein synthesis in all cells. Here, we establish a kinetic framework for the 50S modified fragment reaction that makes it possible to measure the kinetic effects that result from isotopic substitution in either the A or P site of the ribosome. This simplified peptidyl transferase assay follows a rapid equilibrium random mechanism in which the reverse reaction is nonexistent and the forward commitment is negligible. A normal effect (1.009) is observed for N-15 substitution of the incoming nucleophile at both low and high pH. This suggests that the first irreversible step is the formation of the tetrahedral intermediate. The observation of a normal isotope effect that does not change as a function of pH suggests that the ribosome promotes peptide bond formation by a mechanism that differs in its details from an uncatalyzed aminolysis reaction in solution. This implies that the ribosome contributes chemically to catalysis of peptide bond formation.
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收藏
页码:4018 / 4027
页数:10
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