Glutamate 270 plays an essential role in K(+)-activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase.

•The role of a conserved residue (E270) of 3-isopropylmalate dehydrogenase has been determined.•The mutant E270A exhibits largely reduced catalytic activity and negligible activation by K+.•QM/MM calculations predicted an increase in the activation energy of catalysis upon the mutation.•The X-ray structure of the E270A mutant exhibited a bound water molecule in the place of K+.•SAXS and FRET experiments revealed the role of E270 in domain-closure required by the function.