title: 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.
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117 Budapest, Hungary.
authors.forename: Éva
authors.initials: É
authors.lastname: Gráczer
authors.affiliation: Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117 Budapest, Hungary.
authors.forename: Anna
authors.initials: A
authors.lastname: Palló
authors.affiliation: Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Gellért Square 4., H-1111 Budapest, Hungary.
authors.forename: Julianna
authors.initials: J
authors.lastname: Oláh
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117 Budapest, Hungary.
authors.forename: Tamás
authors.initials: T
authors.lastname: Szimler
authors.affiliation: European Molecular Biology Laboratory, Hamburg Outstation, Notkestrasse 85, 22603 Hamburg, Germany.
authors.forename: Petr V
authors.initials: PV
authors.lastname: Konarev
authors.affiliation: European Molecular Biology Laboratory, Hamburg Outstation, Notkestrasse 85, 22603 Hamburg, Germany.
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.affiliation: Universitá degli Studi di Parma, Dipartimento di Bioscienze, Viale G.P. Usberti 23/A, I-43100 Parma, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Merli
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117 Budapest, Hungary.
authors.forename: Péter
authors.initials: P
authors.lastname: Závodszky
authors.affiliation: Helmholtz-Zentrum Berlin für Materialien und Energie, Macromolecular Crystallography (HZB-MX), Albert-Einstein-Str. 15, D-12489 Berlin, Germany.
authors.forename: Manfred S
authors.initials: MS
authors.lastname: Weiss
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117 Budapest, Hungary. Electronic address: vas.maria@ttk.mta.hu.
authors.forename: Mária
authors.initials: M
authors.lastname: Vas
mesh_terms: D050539:3-Isopropylmalate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D018698:Glutamic Acid; D050539:3-Isopropylmalate Dehydrogenase
keywords: Activation by K(+); Fluorescence resonance energy transfer; Isopropylmalate dehydrogenase; Site-directed mutagenesis; Small angle X-ray scattering; X-ray crystallography