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

Hiller, David A.; Singh, Vipender; Zhong, Minghong; Strobel, Scott A.

doi:10.1038/nature10248

Cited by 77 publications

(96 citation statements)

References 31 publications

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“…Both models are consistent with the experimental data available so far, including the pH/rate profiles for different substrates, mutational analysis, and KIE and KSIE measurements (Katunin et al 2002;Youngman et al 2004;Beringer and Rodnina 2007;Lang et al 2008;Wohlgemuth et al 2008;Hiller et al 2011;Kuhlenkoetter et al 2011;Zaher et al 2011). Both models suggest that the proton from the attacking amine is transferred to the leaving group through the 2 ′ -OH of the P-site tRNA A76; however, the impact of L27 and the exact path of proton transfer is different (we note that at a time when the proton shuttle model was suggested, the positions of the water molecules in the catalytic site were not known with precision).…”

Section: Role Of L27 In the Ptcsupporting

confidence: 72%

Activities of the peptidyl transferase center of ribosomes lacking protein L27

Maracci

Wohlgemuth

Rodnina

2015

RNA

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The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A-and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally.Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome.

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Section: Role Of L27 In the Ptcsupporting

confidence: 72%

Activities of the peptidyl transferase center of ribosomes lacking protein L27

Maracci

Wohlgemuth

Rodnina

2015

RNA

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“…Recent KIE and computational results by Hiller et al (37) suggest that in the ribosome active site, the second deprotonation occurs concomitant with C-N bond formation. These examples illustrate that interactions with enzyme active site residues can subtly alter the transition state in a manner reflecting, in part, the nature of catalytic modes that are used.…”

Section: Simulations and Qm Calculations To Model The Mechanism Andmentioning

confidence: 99%

Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2′- O -transphosphorylation

Gu¹,

Zhang²,

Wong

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

125

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Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2′-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/ base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the 18 O KIEs on the 2′O nucleophile, 5′O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium-or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens. Quantum mechanical calculations consistent with the experimental KIEs indicate that expulsion of the 5′O remains an integral feature of the rate-limiting step both on and off the enzyme. Electrostatic interactions with positively charged amino acid site chains (His12/ Lys41), together with proton transfer from His119, render departure of the 5′O less advanced compared with the solution reaction and stabilize charge buildup in the transition state. The ability to obtain a chemically detailed description of 2′-O-transphosphorylation transition states provides an opportunity to advance our understanding of biological catalysis significantly by determining how the catalytic modes and active site environments of phosphoryl transferases influence transition state structure. E nzymes achieve powerful rate enhancements by providing multiple catalytic modes to stabilize reaction transition states, including electrostatic interactions and proton transfer (1). For RNA 2′-O-transphosphorylation reactions, interactions with acid, base, or metal ion catalysts in solution can influence transition state structure (2). Understanding biological catalysis therefore requires knowledge of chemical mechanisms, transition state structure, and transition state interactions for both enzymatic and nonenzymatic RNA cleavage reactions. Comparisons of enzymatic and nonenzymatic catalysis can provide information on which catalytic modes are used by enzymes and whether unique features of the active site environment may alter the transition state charge distribution (3).RNA undergoes two competing transesterification reactions in solution: isomerization to a 2′,5′-phosphodiester and 2′-Otransphosphorylation to yield a cyclic 2′,3′-phosphodiester with concomitant release of the 5′O-nucleoside (4, 5). Reactions catalyzed by acid and by buffers yield both isomerization and cleavage products and proceed via a pentacoordinated phosphorane intermediate formed by an attack of the 2′OH at the adja...

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“…The mechanism of this catalyzed reaction originally proposed by Nissen et al in 2000 (1) involved favorable orientation of peptide precursors, acid-base catalysis and transition-state stabilization, and the altered pK a of the functional groups of the precursors caused by the reaction environment provided by the active site; such pK a shifts had previously been seen in the active sites of other proteins (2). Since then, the original mechanism has been contested (3,4). The acknowledged mechanistic function of the ribosome's active site is its ability to bring the precursors in close proximity and orient them for reaction, with further mechanistic details remaining unresolved (4).…”

mentioning

confidence: 99%

In situ observation of peptide bond formation at the water–air interface

Griffith

Vaida

2012

Proc. Natl. Acad. Sci. U.S.A.

146

155

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We report unambiguous spectroscopic evidence of peptide bond formation at the air-water interface, yielding a possible mechanism providing insight into the formation of modern ribosomal peptide bonds, and a means for the emergence of peptides on early Earth. Protein synthesis in aqueous environments, facilitated by sequential amino acid condensation forming peptides, is a ubiquitous process in modern biology, and a fundamental reaction necessary in prebiotic chemistry. Such reactions, however, are condensation reactions, requiring the elimination of a water molecule for every peptide bond formed, and are thus unfavorable in aqueous environments both from a thermodynamic and kinetic point of view. We use the hydrophobic environment of the air-water interface as a favorable venue for peptide bond synthesis, and demonstrate the occurrence of this chemistry with in situ techniques using Langmuir-trough methods and infrared reflection absorption spectroscopy. Leucine ethyl ester (a small amino acid ester) first partitions to the water surface, then coordinates with Cu 2þ ions at the interface, and subsequently undergoes a condensation reaction selectively forming peptide bonds at the air-water interface.

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

Cited by 77 publications

References 31 publications

Activities of the peptidyl transferase center of ribosomes lacking protein L27

Activities of the peptidyl transferase center of ribosomes lacking protein L27

Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2′- O -transphosphorylation

In situ observation of peptide bond formation at the water–air interface

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