Abstract
Allosteric activators are generally believed to shift the equilibrium distribution of enzyme conformations to favor a catalytically productive structure; the kinetics of conformational exchange is seldom addressed. Several observations suggested that the usual allosteric mechanism might not apply to the activation of IMP dehydrogenase (IMPDH) by monovalent cations. Therefore, we investigated the mechanism of K+ activation in IMPDH by delineating the kinetic mechanism in the absence of monovalent cations. Surprisingly, the K+ dependence of kcat derives from the rate of flap closure, which increases by ≥65-fold in the presence of K+. We performed both alchemical free energy simulations and potential of mean force calculations using the orthogonal space random walk strategy to computationally analyze how K+ accelerates this conformational change. The simulations recapitulate the preference of IMPDH for K+, validating the computational models. When K+ is replaced with a dummy ion, the residues of the K+ binding site relax into ordered secondary structure, creating a barrier to conformational exchange. K+ mobilizes these residues by providing alternate interactions for the main chain carbonyls. Potential of mean force calculations indicate that K+ changes the shape of the energy well, shrinking the reaction coordinate by shifting the closed conformation toward the open state. This work suggests that allosteric regulation can be under kinetic as well as thermodynamic control.