Kinetic Isotope Effects in Enzymes

Abstract

Kinetic isotope effects (KIEs) have progressed to become one of the most useful analytical techniques to study enzymatic reactions and can answer a variety of questions ranging from the basic mechanism of a reaction to the structure of a transition state (TS) and the role of quantum mechanical tunneling. Here, we briefly discuss the development of the theory behind KIEs with an emphasis on how it guides the interpretation of experimental data. We then present some of the instrumentation and techniques commonly used for KIE experiments, followed by a number of different examples from the enzymology literature wherein KIEs have been used to probe chemical transformations, with an emphasis on the effects of quantum mechanical tunneling on KIEs.

Mg2+ Binds to the Surface of Thymidylate Synthase and Affects Hydride Transfer at the Interior Active Site

“…We measured the observed KIEs on the second-order rate constant $k_{cat}∕K_M^{{\mathrm{CH}}_4{\mathrm{normalH}}_4\mathrm{fol}}$ (simplified as k cat /K M hereafter), 51,59 and used Northrop's method to extract the intrinsic KIEs as described before. 26 In the absence of Mg 2+ , the observed KIEs are similar to the intrinsic values (Figure 4A), implying that the hydride transfer is also (at least partially) rate-limiting for k cat / K M .…”

Atom Tunneling in Chemistry

“…Thef ollowing approximate criteria have been proposed for H/D KIEs: [20] ad ifference in the activation energy E A (D)ÀE A (H) > 5.0 kJ mol À1 and ar atio of the preexponential factors of A(H)/A(D) < 0.7. Other criteria, such as Swain-Schaad exponents, [21] despite being frequently used, [10,[22][23][24][25][26] were shown to not always be suitable as indicators for tunneling. [20,27,28] Te chniques to measure KIEs, especially in biochemical systems,are reviewed elsewhere.…”

Relationship of Femtosecond–Picosecond Dynamics to Enzyme-Catalyzed H-Transfer

“…The prevailing model of reactivity, then, is an extension of the Marcus theory of electron tunneling, 16 where the tunneling particle is a hydrogen rather than an electron. This model, which has been described under different names (Marcus-like models, 17 environmentally coupled tunneling, 18 full-tunneling model, 19 vibrationally enhanced tunneling, 20 etc. ), proposes a mechanism where heavy atom reorganization leads to a “Tunneling Ready State” (TRS) in which the quantum states of the hydrogen in the reactant and product wells are degenerate, and, thus, the hydrogen can transfer to products via tunneling.…”

“…non-statistical dynamics. 2, 17, 19, 25, 26 This imprecise language subsequently led some to believe that a controversy exists over whether true non-equilibrium dynamics contribute to enzyme-catalyzed reactions. 27, 28 Although several such proposals do exist, 2 the Marcus-like models do not require and thus do not invoke non-equilibrium dynamics.…”