Catalytic Role of Metal Ion in the Selection of Competing Reaction Paths: A First Principles Molecular Dynamics Study of the Enzymatic Reaction in Ribozyme Mauro Boero,* ,†,‡ Kiyoyuki Terakura, ‡,§ and Masaru Tateno ‡ Contribution from the Angstrom Technology Partnership, Joint Research Center for Atom Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-0046, Japan, National Institute of AdVanced Industrial Science and Technology, Joint Research Center for Atom Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan, and Research Institute for Computational Sciences, National Institute of AdVanced Industrial Science and Technology, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan Received December 21, 2001 Abstract: By using finite temperature first principles molecular dynamics, the mechanism of the enzymatic reaction of ribozyme was investigated for both the anionic and the radical charge states of the modeled RNA fragment. In the case of the anionic system, a pseudorotation and the subsequent 3′ f 2′ migration occur in a vacuum, rather than the self-cleavage of the phosphodiester. On the other hand, when either a divalent metal ion (Mg 2+ ) catalyst or the continuous hydrogen bond network of the solvent is present, the reaction path of the anionic species changes dramatically, going toward the transesterification channel. In a radical system, the transesterification can occur without a metal catalyst, as a consequence of the displacement of a hole (empty electronic state) along the reaction path. Thus, the present analysis suggests that a metal ion might be essential not only in lowering the activation barrier but also in selecting the reaction path among those corresponding to possible different charge states of the intermediate structure in vivo. Furthermore, simulation of the anionic species in solution shows that, in the absence of a metal catalyst, water molecules cooperate with the proton transfer via a proton wire mechanism and the hydrogen bond network plays a crucial role in preventing pseudorotations. On the other hand, when a metal cation is present in the vicinity of the site where the nucleophilic attack occurs, the hydrogen bond network is interrupted and detachment of the proton, enhanced by the catalyst, does not give rise to any proton- transfer process. Introduction Since catalytic RNA molecules (ribozymes) were discovered about 20 years ago, they have gained enormous interest in molecular biology and medical science. The main reason is the fact that ribozymes can be engineered to cleave other target RNA molecules. Hence, they are very active agents able to inhibit gene expression and, for this reason, are very promising candidates in gene therapy of cancer. 1-6 The fundamental chemical reaction operated by ribozymes is hydrolysis of the RNA phosphodiester, resulting in the cleavage of RNA at a particular target site (transesterification). The main steps of the transesterification, according to the most accredited reaction pathway, are summarized in the lower part of Scheme 1 from (1) to (3). However, changes in the environment, such as the pH of the solution, may favor other competing reaction channels. More specifically, in acidic conditions, the 3′ f 2′ phosphodiester migration reaction 7 is known to occur via a pseudorotation Ψ 8 that brings the system from the intermediate configuration 2 in Scheme 1 to a new orientation as in (4) (refer to the literature 1,9-13 for further details). * To whom correspondence should be addressed. E-mail: mauro.boero@ aist.go.jp. † Angstrom Technology Partnership, Joint Research Center for Atom Technology. ‡ National Institute of Advanced Industrial Science and Technology. § National Institute of Advanced Industrial Science and Technology, Joint Research Center for Atom Technology. (1) (a) Zhou, D.; Taira, K. Chem. ReV. 1998, 98, 991. (b) Takagi, Y.; Warashina, M.; Stec, W. J.; Yoshinari, K.; Taira, K. Nucleic Acids Res. 2001, 29, 1815. (c) Yoshinari, K.; Taira, K. Nucleic Acids Res. 2000, 28, 1730. (2) Perreault, D. M.; Anslyn, E. V. Angew. Chem., Int. Ed. Engl. 1997, 36, 433 and references therein. (3) (a) Buzayan, J. M.; Gerlach, W. L.; Bruening, G. Nature 1986, 323, 349. (b) Prody, G. A.; Bakos, J. T.; Buzayan, J. M.; Schneider, I. R.; Bruening, G. Science 1986, 231, 1577. (c) Hutchins, C. J.; Rathjen, P. D.; Forster, A. C.; Symons, R. H. Nucleic Acids Res. 1986, 14, 3627. (d) Hermann, T.; Auffinger, P.; Westhof, E. Eur. Biophys. J. 1998, 27, 153. (4) (a) Joyce, G. F. Science 2000, 289, 401. (b) Schlutes, E. A.; Bartel, D. P. Science 2000, 289, 448. (5) (a) Santoro, S. W.; Joyce, G. F. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4262. (b) Steitz, T. A.; Steitz, J. A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6498. (c) Zhang, B.; Cech, T. R. Chem. Biol. 1998, 5, 539. (d) Bramlage, B.; Luzi, E.; Eckstein, F. Trends Biotechnol. 1998, 16, 434. (6) Kuwabara, T.; Warashima, M.; Taira, K. Trends Biotechnol. 2000, 18, 462. (7) We adopt the standard notation in which atoms are numbered from 1 to 5 on the ribose ring starting from the base, here replaced by a H atom. (8) (a) Westheimer, F. H. Acc. Chem. Res. 1968, 1, 70. (b) Cramer, C. J. J. Am. Chem. Soc. 1990, 112, 7965. (c) Cramer, C. J.; Gustafson, S. M. J. Am. Chem. Soc. 1993, 115, 9315. (d) Cramer, C. J.; Gustafson, S. M. J. Am. Chem. Soc. 1994, 116, 723. (e) Taira, K. Prog. Synth. Oligonucleotides 1995, II, 1323 (in Japanese). Published on Web 07/03/2002 10.1021/ja017843q CCC: $22.00 © 2002 American Chemical Society J. AM. CHEM. SOC. 2002, 124, 8949-8957 9 8949