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Glutamate 270 plays an essential role in K + ‐activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase
Author(s) -
Gráczer Éva,
Palló Anna,
Oláh Julianna,
Szimler Tamás,
Konarev Petr V.,
Svergun Dmitri I.,
Merli Angelo,
Závodszky Péter,
Weiss Manfred S.,
Vas Mária
Publication year - 2015
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2014.12.005
Subject(s) - thermus thermophilus , chemistry , stereochemistry , mutant , ring (chemistry) , nad+ kinase , dehydrogenase , crystallography , active site , oxidoreductase , enzyme , biochemistry , organic chemistry , escherichia coli , gene
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.