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

MÓNICA R. MONTES; SANTIAGO E. FARAJ; ROLANDO C. ROSSI

La Plata, Buenos Aires

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

Ligand-binding and the effect of small molecules on enzyme activity are topics of increasing interest in life sciences curricula since they are critical for drug rational design and the development of new therapeutic agents. In a regular undergraduate course of chemical biology, when addressing enzyme kinetics, the two limiting cases of induced-fit and conformational selection mechanisms are usually presented to students. In the first case, ligand binding occurs before the conformational change takes place, whereas in the latter the conformational change happens before ligand binding. For the sake of simplicity, most of the work done on this subject assumes rapid‐equilibrium for ligand binding without further evidence.We developed a laboratory exercise that demonstrates that assuming rapid- equilibrium may result in a wrong characterization of a ligand-binding mechanism as an induced fit. Students measure eosin fluorescence change induced by the E1 → E2 conformational transition of the Na,K‐ATPase upon addition of Pi or BeF3−. Students observe that Pi and BeF3−produce opposite behaving dependences of the observed rate constants (kobs) with ligandconcentration. Assuming rapid equilibrium for the binding of both ligands, these results suggest a conformational selection mechanism for Pi and an induced- fit mechanism for the binding of BeF3−. However, if no rapid equilibrium assumptions are invoked, both processes may be explained by a conformational selection mechanism.This experiment serves as the basis for discussing the consequence of not being extremely cautious when invoking approximations about not-very-well-known systems and the importance of deeply understanding chemical models used to classify processes.