Glutamate 270 plays an essential role in K+‐activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase. Issue 2 (10th December 2014)

• Record Type: Journal Article
• Authors: Gráczer, Éva; Palló, Anna; Oláh, Julianna; Szimler, Tamás; Konarev, Petr V.; Svergun, Dmitri I.; Merli, Angelo; Závodszky, Péter; Weiss, Manfred S.; Vas, Mária
• Is Part Of: FEBS letters. Volume 589:Issue 2(2015)
• Page Start: 240
• Page End: 245
• Publication Date: 2014-12-10
• Subjects: IPMDH -- 3-isopropylmalate dehydrogenase (EC 1.1.1.85) -- Tt -- Thermus thermophilus -- IPM -- (2R, 3S)-3-isopropylmalate -- MOPS -- 3-(N-morpholino)-propane-sulphonic acid -- SAXS -- small angle X-ray scattering -- FRET -- fluorescence resonance energy transfer -- Isopropylmalate dehydrogenase -- Activation by K+ -- Site-directed mutagenesis -- X-ray crystallography -- Small angle X-ray scattering -- Fluorescence resonance energy transfer; Biochemistry -- Periodicals; Biophysics -- Periodicals; Molecular biology -- Periodicals; Biochimie -- Périodiques; Biochemistry; Biophysics; Molecular biology; Periodicals; 572.05
• DOI: 10.1016/j.febslet.2014.12.005
• Languages: English
• ISSNs: 0014-5793

Abstract

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. Abstract : 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.