Kinetic and thermodynamic analysis of 9-cis-retinoic acid binding to retinoid X receptor alpha.

The interaction of retinoid X receptor alpha with 9-cis-retinoic acid was studied using stopped-flow fluorescence spectroscopy. Transient kinetic analyses of this interaction suggest a two-step binding mechanism involving a rapid, enthalpically driven pre-equilibrium followed by a slower, entropically driven reaction that may arise from a conformational change within the ligand binding domain of the receptor. The assignment of this kinetic mechanism was supported by agreement between the overall equilibrium constant, Kov, derived from kinetic studies with that determined by equilibrium fluorescence titrations. Although these analyses do not preclude ligand-induced alteration in the oligomerization state of the receptor in solution, the simplest model that can be applied to these data involves the stoichiometric interaction of 9-cis-retinoic acid with retinoid X receptor alpha monomers.