Mechanisms of oxygen activation and coupling to carbon-hydrogen cleavage in taurine alpha-ketoglutarate dioxygenase (TauD) and active-site mutants.

摘要

The alpha-ketoglutarate (alpha-KG) dependent non-heme iron oxygenases use the driving force supplied by the oxidative decarboxylation of alpha-KG to oxidize a so-called prime substrate in numerous biologically, medically and economically important pathways. Taurine alpha-KG dioxygenase (tauD) serves as a model system for this diverse family of enzymes. The coupling of the highly oxidizing Fe(IV)-oxo species generated by the oxidative decarboxylation of alpha-KG to subsequent C-H cleavage chemistry is critical to the function of these enzymes. Herein we seek to elaborate the structural determinants of this coupling and the relationship of the oxygen activation mechanism and the C-H cleavage mechanism using kinetic approaches including kinetic isotope effects and the use of substrate analogs in conjunction with site-directed mutagenesis.;The determination of the coupling of oxygen activation to succinate and sulfite production as a function of active site mutation and substrate deuteration reveals the extent to which these perturbations impact oxygen activation and C-H cleavage. The observation that perturbations which greatly impact C-H cleavage do not necessarily affect oxygen activation, despite the requirement that all substrates be bound prior to any reaction occurs, implies a modular component to these reactions. It has also been noted that under conditions where the Fe(IV)-oxo is uncoupled from substrate C-H cleavage the buffer is oxidized. Further, the extent of this reaction with buffer shows a dependence on the size of packing defect introduced by site-directed mutagenesis and the concentration of buffer. The impact of the packing defect introduced by mutagenesis can be partially complemented by the addition of hydrophobic bulk to a substrate analog of taurine, although the C-H cleavage activity is not restored.;The application of steady-state and pre steady-state kinetics along with observed reaction stoichiometries have allowed the interpretation of the impacts of site-directed mutagenesis and substrate deuteration in the context of the strong distance dependence of quantum mechanical hydrogen transfer. The observation of alternate reactivity of the Fe(IV)-oxo intermediate in the presence of poor substrates and enzymes which cannot facilitate substrate orientation with respect to the Fe(IV)-oxo implies a mechanism for the evolution of new activities and substrate specificities.