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Differential Quantum Tunneling Contributions in Nitroalkane Oxidase Catalyzed and the Uncatalyzed Proton Transfer Reaction

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Differential Quantum Tunneling Contributions in Nitroalkane Oxidase Catalyzed and the Uncatalyzed Proton Transfer Reaction Differential quantum tunneling contributions in nitroalkane oxidase catalyzed and the uncatalyzed proton transfer reaction Dan T. Major,a,b,1 Annie Heroux,c Allen M. Orville,c,1 Michael P. Valley,d Paul F. Fitzpatrick,d,1 and Jiali Gaoa,1 aDepartment of Chemistry, Supercomputing Institute and Digital Technology Center, University of Minnesota, Minneapolis, MN 55455; bDepartment of Chemistry and the Lise Meitner-Minerva Center of Computational Quantum Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel; cDepartment of Biology, Brookhaven National Laboratory, Upton, NY 11973; and dDepartment of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229 Communicated by Vern L. Schramm, Albert Einstein College of Medicine, Bronx, NY, October 6, 2009 (received for review July 27, 2009) The proton transfer reaction between the substrate nitroethane over the uncatalyzed reaction between nitroethane and acetate and Asp-402 catalyzed by nitroalkane oxidase and the uncatalyzed ion in water (7). process in water have been investigated using a path-integral Interestingly, the deuterium kinetic isotope effects (KIEs) are free-energy perturbation method. Although the dominating effect noticeably greater for the enzymatic reaction (9.2) than that in in rate acceleration by the enzyme is the lowering of the quasi- water (7.8) (see refs. 7, 8). Although tantalizing, the relatively classical free energy barrier, nuclear quantum effects also contrib- small difference in the observed KIEs is not sufficient to ute to catalysis in nitroalkane oxidase. In particular, the overall conclude that there is a greater tunneling contribution in the nuclear quantum effects have greater contributions to lowering enzymatic process than that of the uncatalyzed reaction in water. the classical barrier in the enzyme, and there is a larger difference A number of experimental and theoretical studies suggested that in quantum effects between proton and deuteron transfer for the the extent of tunneling was the same in several enzymes as in enzymatic reaction than that in water. Both experiment and model reactions in solution (2, 9, 10), whereas other experiments computation show that primary KIEs are enhanced in the enzyme, showing differential tunneling behaviors may be attributed to and the computed Swain-Schaad exponent for the enzymatic different reaction mechanisms (2, 11–13). Nuclear tunneling reaction is exacerbated relative to that in the absence of the cannot be measured directly, and the best experimental diag- enzyme. In addition, the computed tunneling transmission coeffi- nostic of tunneling is through measurement of kinetic isotope cient is approximately three times greater for the enzyme reaction effects (14). Klinman and coworkers showed that the presence than the uncatalyzed reaction, and the origin of the difference may of tunneling can be revealed indirectly by a potentiated Swain- be attributed to a narrowing effect in the effective potentials for Schaad exponent in the secondary KIEs (2, 15, 16). However, tunneling in the enzyme than that in aqueous solution. computational studies can shed light on the extent of nuclear quantum effects (NQE), including both zero-point energies and PI-FEP/UM simulations ͉ enzyme catalysis ͉ kinetic isotope effects ͉ tunneling contributions (3, 17). The proton transfer reaction X-ray structure between nitroethane and Asp-402 catalyzed by NAO (18, 19) provides an excellent opportunity for a comparative study be- lthough the dominant factor in enzyme catalysis is the cause the enzymatic process (Fig. S1) can be directly modeled by the reaction of nitroethane and acetate ion in water (Scheme 1). lowering of the quasiclassical free energy barrier of the A Nitroalkanes represent a prototypical system for the study of enzymatic reaction in comparison with the uncatalyzed process carbon acidity, which exhibit surprisingly slow deprotonation (1–3), quantum mechanical tunneling has been recognized to rates compared with their high acidities (20); this phenomenon also play a role in enzymatic hydrogen transfer reactions (2, 3). has been termed as the nitroalkane anomaly (21, 22). An intriguing, yet unanswered, question is whether enzymes We employ a coupled free-energy perturbation and umbrella- have evolved to enhance tunneling to accelerate the reaction rate sampling simulation technique in Feynman centroid path inte- because quantum effects on rate acceleration are much smaller gral calculations (PI-FEP/UM) to describe the NQE (23). More- than hydrogen bonding and electrostatic stabilization of the over, we adopt a combined quantum mechanical and molecular transition state (1, 4, 5). Nevertheless, a small factor of two in mechanical (QM/MM) method to represent the potential energy rate enhancement can have important physiological impacts. surface (22, 24). Thus, both the electronic structure of the Although it appears straightforward to address this question by reacting system and the nuclear dynamics are treated quantum comparing the enzymatic and the uncatalyzed reaction in solu- mechanically. In our approach (23), we follow a two-step pro- tion, the difficulty is to design a model system that mimics exactly cedure (25) in which we first carry out Newtonian molecular the same enzymatic reaction and mechanism. The present study dynamics simulations to determine the classical mechanical examines the structure of nitroalkane oxidase (NAO) complex potential of mean force (PMF) along the reaction coordinate for with nitroethane and kinetic isotope effects at the primary and secondary sites for the enzymatic and the uncatalyzed reaction. The computational findings are consistent with experimental Author contributions: D.T.M., A.M.O., P.F.F., and J.G. designed research; D.T.M., A.H., and data, suggesting that there is a differential tunneling effect for M.P.V. performed research; D.T.M., A.M.O., P.F.F., and J.G. analyzed data; and D.T.M. and the proton transfer reaction in NAO and in water. Analysis of J.G. wrote the paper. tunneling paths reveals that the enzyme reduces both the free The authors declare no conflict of interest. energy of activation and the width of the effective potential, Data deposition: The atomic coordinates for the D402N NAO plus 1-nitroethane complex and corresponding structure factors have been deposited in the Protein Data Bank, resulting in enhanced proton tunneling in the active site. www.pdb.org (PDB ID code 3FCJ). The flavoenzyme NAO catalyzes the conversion of nitroal- 1To whom correspondence may be addressed. E-mail: [email protected], kanes to nitrite and aldehydes or ketones (Fig. S1) (6). The [email protected], fi[email protected], or [email protected]. ␣ -proton abstraction of the small substrate nitroethane by This article contains supporting information online at www.pnas.org/cgi/content/full/ Asp-402 is rate-limiting, which is accelerated by a factor of 109 0911416106/DCSupplemental. 20734–20739 ͉ PNAS ͉ December 8, 2009 ͉ vol. 106 ͉ no. 49 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0911416106 Downloaded by guest on October 1, 2021 available for an enzyme system, and a computational model must be constructed before simulations can be carried out. Thus, we have performed separate experimental and computational in- vestigations of the NAO⅐nitroethane complex and compared the results after each study was completed. Scheme 1. Proton abstraction of nitroethane by acetate ion in water. We used the NAO isosteric D402N mutant to trap the MC for crystallographic analysis because it is not active with neutral the proton transfer reaction between nitroethane and Asp-402. nitroalkanes (34). The 2.4-Å resolution cocrystal structure with Then, atoms that are directly involved in the proton transfer nitroethane demonstrates that the mutation does not signifi- reaction are quantized, and the configurations sampled in mo- cantly perturb the active site. The electron density maps are lecular dynamics simulations are used in path-integral simula- consistent with nitroethane located above the FAD re-face (Fig. 1A). In the crystal structure, the oxygen atoms of the R-NO2 tions by constraining the centroid positions of the quantized Ј particles to the classical coordinates. This double (quantum and moiety form hydrogen bonds with the ribityl 2 -OH group (3.4 classical) averaging procedure is formally exact (23, 25–28), and 3.7 Å). This hydrogen bonding network places the nitroeth- ane, C␣, directly ‘‘below’’ the side chain of Asn-402. Slightly which yields the QM-PMF as a function of the centroid reaction different substrate orientations are observed in each of the four path (29, 30). In PI-FEP/UM, the ratio of the quantum partition subunits in the crystal structure of the D402N nitroethane functions for different isotopes, which yields the KIEs, is ob- complex (Fig. 1B). The variability likely results from a combi- tained by free-energy perturbation from a light isotope mass into nation of real enzyme-substrate binding effects and the modest a heavier one within the same centroid path-integral simulation resolution of the crystal structure. For comparison, the crystal (23), avoiding the difference between two free-energy barriers structures with the substrates 1-nitrohexane or 1-nitrooctane with greater fluctuations than the difference itself for the two demonstrate similar complexes, but with less binding orientation isotopic reactions. Consequently, the PI-FEP/UM method is variability (33). Thus, nitroethane exhibits the most active site unique in that it yields
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