Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (kcat/KM,IPM) for the substrate IPM has an unusual temperature dependence, showing a local minimum at ∼35°C. In search of an explanation, we measured the individual constants kcat and KM,IPM as a function of temperature, and found that the van 't Hoff plot of KM,IPM shows sigmoid-like transition in the 20-40°C temperature range. By means of various measurements including hydrogen-deuterium exchange and fluorescence resonance energy transfer, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperatures >30°C. The thermodynamic parameters of ligand binding determined by isothermal titration calorimetry as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. Because the binding of IPM is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with IPM binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency. © 2009 by the Biophysical Society.
Hajdú, I., Szilágyi, A., Kardos, J., & Závodszky, P. (2009). A link between hinge-bending domain motions and the temperature dependence of catalysis in 3-isopropylmalate dehydrogenase. Biophysical Journal, 96(12), 5003–5012. https://doi.org/10.1016/j.bpj.2009.04.014