Global stability of an α-ketoglutarate-dependent dioxygenase (TauD) and its related complexes.

BACKGROUND

TauD is a nonheme iron(II) and α-ketoglutarate (αKG) dependent dioxygenase, and a member of a broader family of enzymes that oxidatively decarboxylate αKG to succinate and carbon dioxide thereby activating O to perform a range of oxidation reactions. However before O activation can occur, these enzymes bind both substrate and cofactor in an effective manner. Here the thermodynamics associated with substrate and cofactor binding to FeTauD are explored.

METHODS

Thermal denaturation of TauD and its enzyme-taurine, enzyme-αKG, and enzyme-taurine-αKG complexes are explored using circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC).

RESULTS

Taurine binding is endothermic (+26kcal/mol) and entropically driven that includes burial of hydrophobic surfaces to close the lid domain. Binding of αKG is enthalpically favorable and shows cooperativity with taurine binding, where the change in enthalpy associated with αKG binding (δΔH) increases from -30.1kcal/mol when binding to FeTauD to -65.2kcal/mol when binding to the FeTauD-taurine complex.

CONCLUSIONS

The intermolecular interactions that govern taurine and αKG binding impact the global stability of TauD and its complexes, with clear and dramatic cooperativity between substrate and cofactor.

GENERAL SIGNIFICANCE

Thermal denaturation of TauD and its enzyme-taurine, enzyme-αKG, and enzyme-taurine-αKG complexes each exhibited increased temperature stability over the free enzyme. Through deconvolution of the energetic profiles for all species studied, a thermodynamic cycle was generated that shows significant cooperativity between substrate and cofactor binding which continues to clarity the events leading up O activation.