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.

Glutamate 270 plays an essential role in K(+)-activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase / Éva Gráczer Anna Palló Julianna Oláh Tamás Szimler Petr V. Konarev Dmitri I. Svergun Angelo Merli Péter Závodszky Manfred S. Weiss Mária Vas. - In: FEBS LETTERS. - ISSN 0014-5793. - 589(2015), pp. 240-245. [10.1016/j.febslet.2014.12.005]

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

MERLI, Angelo
2015

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.
Glutamate 270 plays an essential role in K(+)-activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase / Éva Gráczer Anna Palló Julianna Oláh Tamás Szimler Petr V. Konarev Dmitri I. Svergun Angelo Merli Péter Závodszky Manfred S. Weiss Mária Vas. - In: FEBS LETTERS. - ISSN 0014-5793. - 589(2015), pp. 240-245. [10.1016/j.febslet.2014.12.005]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11381/2787454
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