6533b827fe1ef96bd1285bb1

RESEARCH PRODUCT

Glutamate 270 plays an essential role in K+-activation and domain closure ofThermus thermophilusisopropylmalate dehydrogenase

Manfred S. WeissDmitri I. SvergunPetr V. KonarevAnna PallóMária VasAngelo MerliÉVa GráczerJulianna OláhPéter ZávodszkyTamás Szimler

subject

Models MolecularStereochemistry030303 biophysicsMutantBiophysicsGlutamic AcidLarge scale facilities for research with photons neutrons and ionsSmall angle X-ray scatteringDehydrogenaseBiochemistry3-Isopropylmalate Dehydrogenase03 medical and health scienceschemistry.chemical_compoundIsopropylmalate dehydrogenaseFluorescence resonance energy transferStructural BiologyOxidoreductaseGeneticsMolecular BiologyX-ray crystallography030304 developmental biologychemistry.chemical_classificationSite-directed mutagenesis0303 health sciencesNicotinamidebiologyThermus thermophilusActivation by K+Cell BiologyThermus thermophilusbiology.organism_classificationProtein Structure TertiaryMOPSEnzyme ActivationKineticsCrystallographyEnzymechemistryMutationNAD+ kinase

description

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.

yearjournalcountryeditionlanguage
2014-12-10FEBS Letters
https://doi.org/10.1016/j.febslet.2014.12.005