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Role of the active site guanine in the glmS ribozyme self-cleavage mechanism: quantum mechanical/molecular mechanical free energy simulations.

ABSTRACT: The glmS ribozyme catalyzes a self-cleavage reaction at the phosphodiester bond between residues A-1 and G1. This reaction is thought to occur by an acid-base mechanism involving the glucosamine-6-phosphate cofactor and G40 residue. Herein quantum mechanical/molecular mechanical free energy simulations and pKa calculations, as well as experimental measurements of the rate constant for self-cleavage, are utilized to elucidate the mechanism, particularly the role of G40. Our calculations suggest that an external base deprotonates either G40(N1) or possibly A-1(O2'), which would be followed by proton transfer from G40(N1) to A-1(O2'). After this initial deprotonation, A-1(O2') starts attacking the phosphate as a hydroxyl group, which is hydrogen-bonded to deprotonated G40, concurrent with G40(N1) moving closer to the hydroxyl group and directing the in-line attack. Proton transfer from A-1(O2') to G40 is concomitant with attack of the scissile phosphate, followed by the remainder of the cleavage reaction. A mechanism in which an external base does not participate, but rather the proton transfers from A-1(O2') to a nonbridging oxygen during nucleophilic attack, was also considered but deemed to be less likely due to its higher effective free energy barrier. The calculated rate constant for the favored mechanism is in agreement with the experimental rate constant measured at biological Mg(2+) ion concentration. According to these calculations, catalysis is optimal when G40 has an elevated pKa rather than a pKa shifted toward neutrality, although a balance among the pKa's of A-1, G40, and the nonbridging oxygen is essential. These results have general implications, as the hammerhead, hairpin, and twister ribozymes have guanines at a similar position as G40.

SUBMITTER: Zhang S

PROVIDER: S-EPMC4308743 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Role of the active site guanine in the glmS ribozyme self-cleavage mechanism: quantum mechanical/molecular mechanical free energy simulations.

Zhang Sixue S Ganguly Abir A Goyal Puja P Bingaman Jamie L JL Bevilacqua Philip C PC Hammes-Schiffer Sharon S

Journal of the American Chemical Society 20150112 2

The glmS ribozyme catalyzes a self-cleavage reaction at the phosphodiester bond between residues A-1 and G1. This reaction is thought to occur by an acid-base mechanism involving the glucosamine-6-phosphate cofactor and G40 residue. Herein quantum mechanical/molecular mechanical free energy simulations and pKa calculations, as well as experimental measurements of the rate constant for self-cleavage, are utilized to elucidate the mechanism, particularly the role of G40. Our calculations suggest t ...[more]

PMID: 25526516

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Project description:DNA bases can adopt energetically unfavorable tautomeric forms that enable the formation of Watson-Crick-like (WC-like) mispairs, which have been proposed to give rise to spontaneous mutations in DNA and misincorporation errors in DNA replication and translation. Previous NMR and computational studies have indicated that the population of WC-like guanine-thymine (G-T) mispairs depends on the environment, such as the local nucleic acid sequence and solvation. To investigate these environmental effects, herein G-T mispair tautomerization processes are studied computationally in aqueous solution, in A-form and B-form DNA duplexes, and within the active site of a DNA polymerase λ variant. The wobble G-T (wG-T), WC-like G-T*, and WC-like G*-T forms are considered, where * indicates the enol tautomer of the base. The minimum free energy paths for the tautomerization from the wG-T to the WC-like G-T* and from the WC-like G-T* to the WC-like G*-T are computed with mixed quantum mechanical/molecular mechanical (QM/MM) free energy simulations. The reaction free energies and free energy barriers are found to be significantly influenced by the environment. The wG-T→G-T* tautomerization is predicted to be endoergic in aqueous solution and the DNA duplexes but slightly exoergic in the polymerase, with Arg517 and Asn513 providing electrostatic stabilization of G-T*. The G-T*→G*-T tautomerization is also predicted to be slightly more thermodynamically favorable in the polymerase relative to these DNA duplexes. These simulations are consistent with an experimentally driven kinetic misincorporation model suggesting that G-T mispair tautomerization occurs in the ajar polymerase conformation or concertedly with the transition from the ajar to the closed polymerase conformation. Furthermore, the order of the associated two proton transfer reactions is predicted to be different in the polymerase than in aqueous solution and the DNA duplexes. These studies highlight the impact of the environment on the thermodynamics, kinetics, and fundamental mechanisms of G-T mispair tautomerization, which plays a role in a wide range of biochemically important processes.

Dataset Information

Role of the active site guanine in the glmS ribozyme self-cleavage mechanism: quantum mechanical/molecular mechanical free energy simulations.

Publications

Role of the active site guanine in the glmS ribozyme self-cleavage mechanism: quantum mechanical/molecular mechanical free energy simulations.

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