Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase A at low pH

► Thermal denaturation of hexokinase A MG state at pH 2.5 is non-cooperative. ► Ethylene glycol populates non-native and non-cooperative ensemble at pH 2.5. ► Stabilizing polyol osmolytes populate native-like cooperative intermediate state at pH 2.5. ► The cooperative intermediate state binds to the substrate glucose.