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 Mg2+ 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.

Chemical feasibility of the general acid/base mechanism of glmS ribozyme self‐cleavage

“…66 Both the reaction mechanism and the estimated barrier are consistent with our findings. Note that, although both QM/MM studies are based on similar DFT descriptions of the QM core, they differ methodologically in many significant aspects (see the discussion above) and use distinct starting structures.…”

“…This includes the thermodynamics correction for the rare protonation states of G40 and GlcN6P, and a correction for the systematic error associated with the over-polarization in the minimal QM region with electronic embedding (see Methods, Table 2, Figure 2 and Supporting Information). This estimated range is higher than the barrier derived from the experimentally measured k cat of ~93 min −1 at 30 mM MgCl 2 , corresponding to a free energy barrier of 17.3 kcal/mol at 298 K. 26 Notably, while our paper was under revision, another study reported a kinetic measurement at 3 mM MgCl 2 of a k cat of 0.013 ± 0.003 min −1 , corresponding to a barrier of 22.6 ± 0.2 kcal/mol at 300 K. 66 Hammes-Schiffer et al suggested in this study that 3 mM MgCl 2 is closer both to physiological conditions and to the concentration of divalents in the MD simulations. They thus suggested that the new experimental kinetic constant should be used for comparison with the theoretically calculated barriers.…”

“…They thus suggested that the new experimental kinetic constant should be used for comparison with the theoretically calculated barriers. 66 However, such straightforward comparisons are not devoid of potential uncertainties. In particular, divalent ions do not directly participate in glmS ribozyme self-cleavage; they affect catalysis rather indirectly via facilitated GlcN6P binding and more compact RNA folding.…”

“…The TS energy was further corrected by −4.6 kcal/mol to account for the inaccurate QM-MM coupling in the calculations using the minimal QM region. See Supporting Information for explanation of the error estimates. b The range stands for the different rate constants reported for different conditions, namely in the presence of concentrations of 3 mM MgCl 2 66 and 30 mM MgCl 2 26 . …”

Determination of pKa values for deprotonable nucleobases in short model oligonucleotides

“…For comparison, the pK a values of thymine, uracil, and hypoxanthine moieties located in such oligonucleotides were also determined. The results should be useful for providing estimates for local electric charges of interest in ribozyme mechanisms [27], efficient conversion of dsDNA to ssDNA for nanopore-based DNA sequencing [28], and prediction of differential electrophoretic or chromatographic mobilities of single-stranded oligonucleotides under different pH conditions.…”