Theoretical Study of Phosphodiester Hydrolysis in Nucleotide Pyrophosphatase/Phosphodiesterase. Environmental Effects on the Reaction Mechanism Violeta Lo ´ pez-Canut, ‡ Maite Roca, ‡ Juan Bertra ´n, † Vicent Moliner,* ,§,∫ and In ˜aki Tun ˜o ´n* ,‡ Departament de Quı ´mica Fı ´sica, UniVersitat de Vale `ncia, 46100 Burjassot, Spain, Departament de Quı ´mica Fı ´sica i Analı ´tica, UniVersitat Jaume I, 12071 Castello ´n, Spain, Institute of Applied Radiation Chemistry, Technical UniVersity of Lodz, 90-924 Lodz, Poland, and Departament de Quı ´mica; UniVersitat Auto `noma de Barcelona, 08193 Bellaterra, Spain Received October 2, 2009; E-mail: ignacio.tunon@uv.es; moliner@uji.es Abstract: We here present a theoretical study of the alkaline hydrolysis of methyl p-nitrophenyl phosphate (MpNPP - ) in aqueous solution and in the active site of nucleotide pyrophosphatase/phosphodiesterase (NPP). The analysis of our simulations, carried out by means of hybrid quantum mechanics/molecular mechanics (QM/MM) methods, shows that the reaction takes place through different reaction mechanisms depending on the environment. Thus, while in aqueous solution the reaction occurs by means of an A N D N mechanism, the enzymatic process takes place through a D N A N mechanism. In the first case, we found associative transition-state (TS) structures, while in the enzyme TS structures have dissociative character. The reason for this change is rationalized in terms of the very different nature of the electrostatic interactions established in each of the environments: while the aqueous solution reduces the repulsion between the negatively charged reacting fragments, assisting their approach, the NPP active site stabilizes the charge distribution of dissociative TS structures, allowing the reaction to proceed with a significantly reduced free energy cost. Interestingly, the NPP active site is able to accommodate different substrates, and it seems that the nature of the TSs depends on their electronic characteristics. So, in the case of the MpNPP - substrate, the nitro group establishes hydrogen-bond interactions with water molecules and residues found in the outer part of the catalytic site, while the leaving group oxygen atom does not coordinate directly with any of the zinc atoms of the active site. If methyl phenyl phosphate is used as substrate, then the charge on the leaving group is supported to larger extent by the oxygen atom and the phenolate anion can be then coordinated to one of the two zinc atoms present in the active site. 1. Introduction Phosphoester hydrolysis is a fundamental process in biological systems. Many biological molecules contain phosphate and reactions in which phosphorus-oxygen bonds are broken are important for energy flow, signal transduction, and genetic inheritance. 1-8 The kinetic stability of the phosphorus-oxygen bond in aqueous solution imposes the use of enzymes to reach chemical rates compatible with life. 9 In fact, enzymes involved in the catalysis of these reactions, such as kinases, ATPases, and phosphatases, have some of the largest catalytic activities known. 10 In spite of their fundamental importance, the reaction mech- anism of phosphoesters hydrolysis is still a controversial question. In principle, phosphoesters hydrolysis can proceed through different reaction mechanisms. 11 Two different limits can be traced on a More-O’Ferrall-Jencks diagram describing two stepwise mechanisms (see Scheme 1): (i) a dissociative reaction mechanism in which cleavage of the phosphorus leaving group bond precedes the formation of the new phosphorus- nucleophile bond (D N + A N mechanism) or (ii) an associative mechanism in which there is no significant bond cleavage to the leaving group while the new bond to the nucleophile is established (A N + D N mechanism). Concerted processes can also be traced on the diagram as a diagonal from reactants to products, and depending on the synchronicity of the breaking and forming bonds (i.e., deviations from the diagonal), the mechanism would be classified as dissociative or associative. ‡ Universitat de Vale `ncia. † Universitat Auto `noma de Barcelona. § Universitat Jaume I. ∫ Technical University of Lodz. (1) Vetter, I. R.; Wittinghofer, A. Q. ReV. Biophys. 1999, 32, 1–56. (2) Cleland, W. W.; Hengge, A. C. Chem. ReV. 2006, 106, 3252–3278. (3) Westheimer, F. H. Science 1987, 235, 1173–1178. (4) Benkovic, S. J., Schray, K. J. In The Enzymes, Vol. VIII; Boyer, P. D. Ed; Academic Press: New York, 1973, pp. 201-238. (5) Kirby, J. A.; Warren, S. G. The Organic Chemistry of Phosphorous; Elsevier: Amsterdam, 1967. (6) Westheimer, F. H. Chem. ReV. 1981, 81, 313–326. (7) Mildvan, A. S. AdV. Enzymol. Relat. Areas Mol. Biol. 1979, 49, 103– 126. (8) Cox, J. R., Jr.; Ramsay, O. B. Chem. ReV. 1964, 64, 317–352. (9) Lad, C.; Williams, N. H.; Wolfenden, R. Proc. Nat. Acad. Sci. U.S.A. 2003, 100, 5607–5610. (10) Zalatan, J. G.; Herschlag, D. J. Am. Chem. Soc. 2006, 128, 1293– 1303. (11) Zalatan, J. G.; Fenn, T. D.; Brunger, A. T.; Herschlag, D. Biochemistry 2006, 45, 9788–9803. Published on Web 04/29/2010 10.1021/ja908391v  2010 American Chemical Society J. AM. CHEM. SOC. 2010, 132, 6955–6963 9 6955