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

Abstract

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 3-4 kcal/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.

Keywords: Activation by K(+); Fluorescence resonance energy transfer; Isopropylmalate dehydrogenase; Site-directed mutagenesis; Small angle X-ray scattering; X-ray crystallography.