Article

A two-step chemical mechanism for ribosome-catalysed peptide bond formation.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Nature (Impact Factor: 42.35). 08/2011; 476(7359):236-9. DOI: 10.1038/nature10248
Source: PubMed

ABSTRACT The chemical step of natural protein synthesis, peptide bond formation, is catalysed by the large subunit of the ribosome. Crystal structures have shown that the active site for peptide bond formation is composed entirely of RNA. Recent work has focused on how an RNA active site is able to catalyse this fundamental biological reaction at a suitable rate for protein synthesis. On the basis of the absence of important ribosomal functional groups, lack of a dependence on pH, and the dominant contribution of entropy to catalysis, it has been suggested that the role of the ribosome is limited to bringing the substrates into close proximity. Alternatively, the importance of the 2'-hydroxyl of the peptidyl-transfer RNA and a Brønsted coefficient near zero have been taken as evidence that the ribosome coordinates a proton-transfer network. Here we report the transition state of peptide bond formation, based on analysis of the kinetic isotope effect at five positions within the reaction centre of a peptidyl-transfer RNA mimic. Our results indicate that in contrast to the uncatalysed reaction, formation of the tetrahedral intermediate and proton transfer from the nucleophilic nitrogen both occur in the rate-limiting step. Unlike in previous proposals, the reaction is not fully concerted; instead, breakdown of the tetrahedral intermediate occurs in a separate fast step. This suggests that in addition to substrate positioning, the ribosome is contributing to chemical catalysis by changing the rate-limiting transition state.

0 Bookmarks
 · 
223 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Studies of RNA recognition and catalysis typically involve measurement of rate constants for reactions of individual RNA sequence variants by fitting changes in substrate or product concentration to exponential or linear functions. A complementary approach is determination of relative rate constants by internal competition, which involves quantifying the time dependent changes in substrate or product ratios in reactions containing multiple substrates. Here, we review approaches for determining of relative rate constants by analysis of both substrate and product ratios and illustrate their application using the in vitro processing of precursor tRNA by ribonuclease P as a model system. The presence of inactive substrate populations is a common complicating factor in analysis of reactions involving RNA substrates, and approaches for quantitative correction of observed rate constants for these effects are illustrated. These results together with recent applications in the literature indicate that internal competition offers an alternate method for analyzing RNA processing kinetics using standard molecular biology methods that directly quantifies substrate specificity and may be extended to a range of applications.
    Analytical Biochemistry 08/2014; DOI:10.1016/j.ab.2014.08.022 · 2.31 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Body tissues are generally 15N-enriched over the diet, with a discrimination factor (Δ15N) that varies among tissues and individuals as a function of their nutritional and physiopathological condition. However, both 15N bioaccumulation and intra- and inter-individual Δ15N variations are still poorly understood, so that theoretical models are required to understand their underlying mechanisms. Using experimental Δ15N measurements in rats, we developed a multi-compartmental model that provides the first detailed representation of the complex functioning of the body's Δ15N system, by explicitly linking the sizes and Δ15N values of 21 nitrogen pools to the rates and isotope effects of 49 nitrogen metabolic fluxes. We have shown that (i) besides urea production, several metabolic pathways (e.g., protein synthesis, amino acid intracellular metabolism, urea recycling and intestinal absorption or secretion) are most probably associated with isotope fractionation and together contribute to 15N accumulation in tissues, (ii) the Δ15N of a tissue at steady-state is not affected by variations of its P turnover rate, but can vary according to the relative orientation of tissue free amino acids towards oxidation vs. protein synthesis, (iii) at the whole-body level, Δ15N variations result from variations in the body partitioning of nitrogen fluxes (e.g., urea production, urea recycling and amino acid exchanges), with or without changes in nitrogen balance, (iv) any deviation from the optimal amino acid intake, in terms of both quality and quantity, causes a global rise in tissue Δ15N, and (v) Δ15N variations differ between tissues depending on the metabolic changes involved, which can therefore be identified using simultaneous multi-tissue Δ15N measurements. This work provides proof of concept that Δ15N measurements constitute a new promising tool to investigate how metabolic fluxes are nutritionally or physiopathologically reorganized or altered. The existence of such natural and interpretable isotopic biomarkers promises interesting applications in nutrition and health.
    PLoS Computational Biology 10/2014; 10(10):e1003865. DOI:10.1371/journal.pcbi.1003865 · 4.83 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Within the quantum theory of atoms in molecules (QTAIM) framework we present a quantum topology phase diagram (QTPD) using the Poincaré–Hopf relation of a total of 17 all new QTAIM topologies of the cis- and trans-isomers of the cyclic contryphan-Sm peptide. The resultant QTPD consists of separate regions for the cis- and trans-isomers that only overlap for topologies associated with the lowest energy minima of the cis- and trans-isomers. We determine the QTAIM topologies of 29 “missing” isomers. A new, contracted formulation of the QTPD is presented, this contracted formulation includes the interamino acid bond critical points (BCPs) that link together the amino acid units, the disulphide bridge “pivot” BCP and side chain bonding interactions. The seven interamino acid BCPs linking the amino acid units coincide with the so-called peptide backbone, the conventional qualitative approach to reduce the complexity of the peptide. We expand the interpretation of ellipticity to include the associated eigenvectors and find that higher values of the ellipticity ɛ are associated with a greater preference to conserve folding states. We quantify previous qualitative findings that suggested the disulfide bond is central to the folding behavior of the cyclic contryphan-Sm peptide and why the cis-isomer is the major form of the cyclic contryphan-Sm peptide. © 2014 Wiley Periodicals, Inc.
    International Journal of Quantum Chemistry 09/2014; 114(24). DOI:10.1002/qua.24784 · 1.17 Impact Factor

Preview

Download
6 Downloads
Available from