| Résumé | A general analytical model is introduced for the analysis of temperature-dependent rate constants in enzymatic hydrogen and deuterium transfer reactions. It exploits the relationship between kinetic isotope effects (KIEs) and their temperature dependence in tunneling reaction to derive criteria that indicate whether a data set can be assigned to one rate-determining tunneling step in the enzymatic reaction sequence. If so, the model evaluates the relative contributions of the tunneling mode and supporting skeletal modes to transfer and provides information on these modes. Recently reported kinetic data on proton transfer in linoleic acid catalyzed by soybean lipoxygenase-1 (SLO1) and six mutants are analyzed by the model, which includes two oscillators, one representing proton motion relative to the skeletal framework and the other the supporting framework motions. It is concluded that most but not all components of this data set can be assigned to a single tunneling step, possible exceptions being associated with the highest observed rates, in agreement with evolutionary expectations. This still allows a complete analysis, namely in terms of the deuteron rate constants. The analysis evaluates how the contributions to proton transfer of the two modes and the electronic term depend on transfer distances in the enzyme and its mutants. It also provides rationalizations for several apparent anomalies, such as the vanishing of the activation energy for some mutants and the observed invariance of the KIE among mutants with very different activity. |
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