Differential quantum tunneling contributions in nitroalkane oxidase catalyzed and the uncatalyzed proton transfer reaction

Abstract

The proton transfer reaction between the substrate nitroethane and Asp-402 catalyzed by nitroalkane oxidase and the uncatalyzed process in water have been investigated using a path-integral free-energy perturbation method. Although the dominating effect in rate acceleration by the enzyme is the lowering of the quasiclassical free energy barrier, nuclear quantum effects also contribute to catalysis in nitroalkane oxidase. In particular, the overall nuclear quantum effects have greater contributions to lowering the classical barrier in the enzyme, and there is a larger difference in quantum effects between proton and deuteron transfer for the enzymatic reaction than that in water. Both experiment and computation show that primary KIEs are enhanced in the enzyme, and the computed Swain-Schaad exponent for the enzymatic reaction is exacerbated relative to that in the absence of the enzyme. In addition, the computed tunneling transmission coefficient is approximately three times greater for the enzyme reaction than the uncatalyzed reaction, and the origin of the difference may be attributed to a narrowing effect in the effective potentials for tunneling in the enzyme than that in aqueous solution.PI-FEP/UM simulations ͉ enzyme catalysis ͉ kinetic isotope effects ͉ X-ray structure A lthough the dominant factor in enzyme catalysis is the lowering of the quasiclassical free energy barrier of the enzymatic reaction in comparison with the uncatalyzed process (1-3), quantum mechanical tunneling has been recognized to also play a role in enzymatic hydrogen transfer reactions (2, 3). An intriguing, yet unanswered, question is whether enzymes have evolved to enhance tunneling to accelerate the reaction rate because quantum effects on rate acceleration are much smaller than hydrogen bonding and electrostatic stabilization of the transition state (1, 4, 5). Nevertheless, a small factor of two in rate enhancement can have important physiological impacts. Although it appears straightforward to address this question by comparing the enzymatic and the uncatalyzed reaction in solution, the difficulty is to design a model system that mimics exactly the same enzymatic reaction and mechanism. The present study examines the structure of nitroalkane oxidase (NAO) complex 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 data, suggesting that there is a differential tunneling effect for the proton transfer reaction in NAO and in water. Analysis of tunneling paths reveals that the enzyme reduces both the free energy of activation and the width of the effective potential, resulting in enhanced proton tunneling in the active site.The flavoenzyme NAO catalyzes the conversion of nitroalkanes to nitrite and aldehydes or ketones (Fig. S1) (6). The ␣-proton abstraction of the small substrate nitroethane by Asp-402 is rate-limiting, which is accelerated by a factor of 10 9 over the uncatalyzed reaction between n...

Unraveling the role of protein dynamics in dihydrofolate reductase catalysis

“…Isotopic substitution of substrates or cofactors has provided strong evidence for quantum tunneling in enzyme reactions. The temperature and pressure dependences of experimentally observed reaction rates and kinetic isotope effects have been interpreted to be a consequence of protein motions on the picoto femtosecond timescale that reduce the width and/or height of the potential energy barrier along the chemical reaction coordinate (1)(2)(3)(4)43). Others have postulated that millisecond conformational fluctuations may also be involved in driving the chemical step of the reaction (22).…”

Perspective: Defining and quantifying the role of dynamics in enzyme catalysis

“…144 studied differential NQM contributions in the catalyzed and uncatalyzed proton transfer reactions of nitroalkane oxidase, and calculated an 8.3 kcal/mol barrier reduction in the enzyme catalyzed reaction compared to the corresponding uncatalyzed reaction in solution (see Table 1 of Ref. 144), which is equivalent to a 10 8 -fold rate acceleration. Of this tremendous barrier reduction, only 0.6 kcal/mol was calculated to be due to tunneling effects (<10-fold) and thus although very slightly larger tunneling contributions were observed in the enzyme than in solution, the contribution to the rate-acceleration is still negligible.…”

Multiscale Quantum‐Classical Simulations of Enzymes

“…[4] This in turn may be associated with the considerably greater cost of solvent reorganization on moving from the reactant state (RS) to the TS in the nonenzymatic reaction compared with that in the enzyme reaction, where the active site is preorganized. [6] Numerous additional catalytic effects, [7] such as RS destabilization, desolvation, [7] covalent bonding, [8] quantum mechanical (QM) tunneling, [9][10][11] and enzyme dynamics, [12,13] have been suggested.…”