Published on Web 10/05/2006
r-Secondary Isotope Effects as Probes of “Tunneling-Ready”
Configurations in Enzymatic H-Tunneling: Insight from
Environmentally Coupled Tunneling Models
Christopher R. Pudney,†,‡ Sam Hay,†,‡ Michael J. Sutcliffe,†,§ and
Nigel S. Scrutton*,†,‡
Contribution from the Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, and
School of Chemical Engineering and Analytical Science, UniVersity of Manchester, 131 Princess
Street, Manchester M1 7ND, United Kingdom
Received March 2, 2006; Revised Manuscript Received August 30, 2006; E-mail: nigel.scrutton@manchester.ac.uk
Abstract: Using R-secondary kinetic isotope effects (2° KIEs) in conjunction with primary (1°) KIEs, we
have investigated the mechanism of environmentally coupled hydrogen tunneling in the reductive half-
reactions of two homologous flavoenzymes, morphinone reductase (MR) and pentaerythritol tetranitrate
reductase (PETNR). We find exalted 2° KIEs (1.17-1.18) for both enzymes, consistent with hydrogen
tunneling. These 2° KIEs, unlike 1° KIEs, are independent of promoting motionssa nonequilibrium pre-
organization of cofactor and active site residues that is required to bring the reactants into a “tunneling-
ready” configuration. That these 2° KIEs are identical suggests the geometries of the “tunneling-ready”
configurations in both enzymes are indistinguishable, despite the fact that MR, but not PETNR, has a
clearly temperature-dependent 1° KIE. The work emphasizes the benefit of combining studies of 1° and 2°
KIEs to report on pre-organization and local geometries within the context of contemporary environmentally
coupled frameworks for H-tunneling.
tunneling by barrier compression in some enzyme systems4,7 is
controversial.8,9 This fast compressive motion is accommodated
Introduction
Hydrogen tunneling is increasingly being implicated in
enzyme reactions.1,2 Experimental studies have emphasized the
inadequacies of the Bell tunnel correction model of semiclassical
transition state theory (TST)3 and the importance of environ-
mental reorganization to the tunneling reaction.1 Physical
models2 that account for experimental criteria for H-tunneling4,5
have emerged, and computational analyses have furnished
important new insight at the atomic level.6 However, a require-
ment for fast (subpicosecond) “promoting motions” that enhance
in physical models of enzymatic H-tunneling2 and inferred from
some experimental4,7 and computational6b,c,f analyses. However,
computational8 and solution kinetic studies9 have also argued
against the need for fast compressive motion along the H-transfer
coordinate.
The analysis of kinetic isotope effects (KIEs) has been at the
heart of experimental descriptions of H-tunneling in enzyme
systems. In particular, the temperature (in)dependence of
primary (1°) KIEs has contributed to the understanding and
formulation of modern, environmentally coupled models of
H-tunneling in enzyme systems.4,5 The reductive half-reactions
of two homologous enzymes, morphinone reductase (MR) from
Pseudomonas putida M1010,11 and pentaerythritol tetranitrate
reductase (PETNR) from Enterobacter cloacae, have been
shown to exhibit temperature-dependent and temperature-
independent (at least within the limit of experimental detection)
1° KIEs, respectively.10 In the context of the environmentally
coupled model of H-tunneling, the temperature dependence of
† Manchester Interdisciplinary Biocentre.
‡ Faculty of Life Sciences.
§ School of Chemical Engineering and Analytical Science.
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10.1021/ja0614619 CCC: $33.50 © 2006 American Chemical Society
J. AM. CHEM. SOC. 2006, 128, 14053-14058
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