Effect of Zn2 + binding and enzyme active site on the transition state for RNA 2′-O-transphosphorylation interpreted through kinetic isotope effects☆

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Highlights

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    KIE measurements suggest that Zn2 + alters the transition state in RNA transphosphorylation.

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    Different Zn2 + binding modes yield distinct KIE signatures.

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    KIEs from one dimetallic binding mode agree best with experimental results.

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    The dimetallic binding mode highly resembles the catalytic mode in RNase A.

Abstract

Divalent metal ions, due to their ability to stabilize high concentrations of negative charge, are important for RNA folding and catalysis. Detailed models derived from the structures and kinetics of enzymes and from computational simulations have been developed. However, in most cases the specific catalytic modes involving metal ions and their mechanistic roles and effects on transition state structures remain controversial. Valuable information about the nature of the transition state is provided by measurement of kinetic isotope effects (KIEs). However, KIEs reflect changes in all bond vibrational modes that differ between the ground state and transition state. QM calculations are therefore essential for developing structural models of the transition state and evaluating mechanistic alternatives. Herein, we present computational models for Zn2 + binding to RNA 2′O-transphosphorylation reaction models that aid in the interpretation of KIE experiments. Different Zn2 + binding modes produce distinct KIE signatures, and one binding mode involving two zinc ions is in close agreement with KIEs measured for non-enzymatic catalysis by Zn2 + aquo ions alone. Interestingly, the KIE signatures in this specific model are also very close to those in RNase A catalysis. These results allow a quantitative connection to be made between experimental KIE measurements and transition state structure and bonding, and provide insight into RNA 2′O-ransphosphorylation reactions catalyzed by metal ions and enzymes. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment.

Introduction

Divalent metal ions play critical roles in RNA folding and catalysis [1], [2], [3], [4], [5], [6], [7], [8]. The ability of divalent ions to stabilize high concentrations of negative charge in transphosphorylation reaction centers via electrostatic interactions, direct coordination or acid-base chemistry empowers them with potential mechanisms to assist in catalysis. However, unraveling the specific role of metal ions is extremely challenging due to the difficulty in discerning the catalytically active metal ion binding mode and its connection with the transition state (TS) structure and bonding [2], [3], [4], which also exists as the major barrier in the investigation of enzyme catalysis mechanisms.

A powerful strategy to resolve mechanistic ambiguity is to rationally design and study simplified model reaction systems using a joint experimental/theoretical approach. Perhaps the most sensitive experimental mechanistic probe is the measurement of kinetic isotope effects (KIEs) that compare the relative reaction rate constants between isotopologues. KIEs arise from subtle quantum effects associated with the changes in structure and bonding that occur in proceeding from the reactant state (RS) to rate-controlling TS [9], [10], [11], [12], [13], [14]. However, meaningful interpretation of KIE measurements requires the use of computational models. Computational modeling of KIEs has been extensively applied to study RNA transphosphorylation catalyzed by enzyme, [15] specifically designed metal catalyst [16], [17] and without catalyst [18], [19], [20]. In a recent work, [21] Zhang et al. measured the primary and secondary kinetic isotope effects for catalysis by Zn2 + ions and by specific base alone, and compared results with preliminary calculations. In the present work, we extend the scope of these calculations to explore 9 distinct, alternative Zn2 + ion binding modes (Fig. 2) within several classes (Scheme 1) in the TS and characterize the resulting KIE signatures. Comparison across different model reactions is also performed and analyzed.

Section snippets

Building a baseline model for un-catalyzed RNA 2′-O-transphosphorylation

In order to understand the effect of Zn2 + binding on TS structure, it is necessary to first characterize the reaction mechanism and TS in the absence of Zn2 +. The transition states for a series of non-enzymatic baseline models (B1–B3) in the absence of Zn2 + are shown in Fig. 1, and their calculated KIEs are compared with experimental values [15] for a UpG dinucleotide in Table 1. As the models progress from the minimal model (B1) to the full dinucleotide (B3), the agreement between the

Conclusion

In conclusion, we explored the effect of different Zn2 + binding modes on the 18O kinetic isotope effects for Zn2 +-catalyzed RNA 2′O-transphosphorylation. Different Zn2 + binding modes yield distinct KIE signatures that can be connected to TS structure and bonding and used to aid in the interpretation of experimental measurements to give insight into mechanism. A unique binding mode was identified as being very closely aligned with recent experimental measurements. This mode involved two zinc

Computational methods

DFT calculations were performed using the B3LYP [24], [25] functional which has been demonstrated to be reliable for zinc complexes [26]. The 6 − 31 + G(d) basis set was used for H, C, N, O and P, while the SDD effective core potential [27] was applied to Zn. Solvation effects were treated with the polarizable continuum model [28] (PCM) using specialized atomic cavity radii for RNA catalysis adopted from previous work [15], [19]. Water solvent with a dielectric constant of 78.4 is used in all PCM

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Acknowledgments

The authors are grateful for financial support provided by the National Institutes of Health (GM062248 to D.M.Y., GM096000 to M.E.H. and AI081987 to J.A.P.). This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number OCI-1053575.

References (29)

  • L.C.S. Melander et al.

    Reaction Rates of Isotopic Molecules

    (1980)
  • A.C. Hengge

    Isotope effects in the study of phosphoryl and sulfuryl transfer reactions

    Acc. Chem. Res.

    (2002)
  • M. Wolfsberg et al.

    Isotope Effects in the Chemical, Geological and Bio Sciences

    (2009)
  • J.K. Lassila et al.

    Biological phosphoryl-transfer reactions: understanding mechanism and catalysis

    Annu. Rev. Biochem.

    (2011)

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    This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment.

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