The mutant E270A of Thermus thermophilus 3-isopropylmalate dehydrogenase exhibits largely reduced (∼1%) catalytic activity and negligible activation by K + compared to the wild-type enzyme. A 3–4kcal/mol increase in the activation energy of the catalysed reaction upon this mutation could also be predicted by QM/MM calculations. In the X-ray structure of the E270A mutant a water molecule was observed to take the place of K + . SAXS and FRET experiments revealed the essential role of E270 in stabilisation of the active domain-closed conformation of the enzyme. In addition, E270 seems to position K + into close proximity of the nicotinamide ring of NAD + and the electron-withdrawing effect of K + may help to polarise the aromatic ring in order to aid the hydride-transfer.