How to distinguish ligand-binding mechanisms: an example of conformational selection disguised as an induced fit

ABSTRACT

This report describes the implementation of a laboratory exercise for an advanced biochemistry or enzyme kinetics class at the undergraduate or graduate level, designed to improve understanding of protein conformational changes associated with the binding of a ligand. Students measure the fluorescence changes induced by the conformational transition of a glycoprotein (the Na,K-ATPase) upon addition of different ligands (Pi and BeF₃⁻) and analyse the results in order to determine the mechanism of the process. The results show that Pi and BeF₃⁻ present opposite effects on the observed rate constants (k_obs) with ligand concentration: k_obs decreases with [Pi] and increases with [BeF₃⁻]. This observation, together with the frequently used assumption that binding occurs under rapid equilibrium, led to propose different models for ligand-induced conformational transitions: a conformational selection for Pi and an induced fit for BeF₃⁻. In this paper, we show that if the rapid-equilibrium approximation for ligand binding is not assumed, a conformational selection mechanism can account for the effects of both ligands. This active-learning exercise serves as the basis for discussing the consequences of not being extremely cautious when invoking approximations about not-very-well-known systems and the importance of a correct understanding of models assigned to chemical processes.

KEYWORDS:

• Protein-ligand interaction
• enzyme kinetics
• Na
• K-ATPase
• induced fit
• conformational selection

Disclosure statement

No potential conflict of interest was reported by the authors.

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Funding

This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica under Grant PICT 2012-2014, 1053; Consejo Nacional de Investigaciones Científicas y Técnicas under Grant PIP 11220150100250CO; and Universidad de Buenos Aires under Grant UBACyT 2014-2017, 20020130100302BA.