Heavy enzymes: Probing fast dynamics in enzyme catalysis by mass modulation

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A central paradigm in biochemistry is that protein function is defined by structure. However, in solution proteins are inherently dynamic molecules, exhibiting motions on timescales ranging from bond stretches through to slow domain motions and normal mode vibrations. An important open question in enzymology remains the role of such dynamics, and whether motions/vibrations on timescales faster than turnover can couple to chemical steps during catalysis. There has been good progress in establishing the role of slower dynamics such as loop opening/closing during enzyme turnover using NMR approaches, but direct evidence for the coupling of faster (sub-nanosecond) dynamics to chemistry remains illusive and controversial and is based largely on the anomalous temperature dependencies of kinetic isotope effects (KIE; e.g. the ratio of rate constants: kH/kD). The role of such fast dynamics remains an important question, as motions on similar timescales to chemistry have the potential to profoundly affect the reaction outcome, and thus offer a means to control (enzyme) reactivity. Stable isotopically-labelled proteins (typically with 2H, 13C and/or 15N) have been exploited as an experimental tool for many years, particularly by the NMR and vibrational spectroscopy communities. The implicit assumption has generally been that isotopic labelling does not significantly perturb protein function. However, it was demonstrated that several isotopically labelled 'heavy' enzymes have measurably slower reaction kinetics. These data were interpreted in terms of the 'Born-Oppenheimer approximation', where increased protein mass (due to labelling) alters bond vibrational frequencies without affecting electrostatic properties of the enzyme. In this case, these results suggest that the lower frequency of (fast) bond vibrations in the 'heavy enzymes' may lead to a reduction in conformational sampling and thus chemical barrier crossing; the rate of reaction is proportional to the rate of barrier crossing. We recently extended the 'heavy enzyme' approach to study vibrational coupling in the Old Yellow enzyme (OYE) pentaerythritol tetranitrate reductase (PETNR). We showed that the temperature dependence of the KIE, which is often used as definitive evidence of protein environmental coupling, is significantly increased in 'heavy' PETNR. This strongly suggests that vibrational coupling can be enhanced by isotopic labelling of proteins. Clearly, the 'heavy enzyme' methodology can be used as a powerful tool to study enzyme coupling and dynamics, but important questions remain. Mass perturbation will affect all vibrations within the protein, so experimental observation of the timescale(s) of any relevant vibrational coupling between protein and chemical coordinate is highly desirable in order to firmly establish the theoretical origin of the 'heavy enzyme' effect. Further, a computational study of a 'heavy' DHFR enzymes suggests that an increased dynamic coupling to the chemical coordinate is detrimental to DHFR catalysis. It is now timely to also consider whether the dynamic coupling of enzyme motions to the chemical coordinate is generally optimised (e.g. by evolution) and thus whether this could be exploited to enhance reactivity or 'drug' enzyme targets.By combining our unique variable temperature and pressure KIE measurements with the 'heavy' enzyme method, we will study the H transfer reactions during both halves of the catalytic cycles of two homologous OYEs, PETNR and morphinone reductase (MR). Selective cofactor and amino acid labelling we will identify (if present) networks of vibrationally-coupled residues, while molecular dynamics simulations and ultrafast spectroscopies will be used to establish the timescales of such coupled vibrations.