title: Dual Role of the Active Site Residues of Thermus thermophilus 3-Isopropylmalate Dehydrogenase: Chemical Catalysis and Domain Closure.
abstract: The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt-IPMDH) have been replaced, one by one, with Ala. A drastic decrease in the kcat value (0.06% compared to that of the wild-type enzyme) has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions (Km values) of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some (1-13%) of the catalytic activity of the wild-type enzyme and do not exhibit appreciable changes in the substrate Km values. The pH dependence of the wild-type enzyme activity (pK = 7.4) is shifted toward higher values for mutants K185A and D241A (pK values of 8.4 and 8.5, respectively). For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics/molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme-substrate complex revealed their additional importance (in particular that of K185, D217, and D241) in stabilizing the domain-closed active conformation. In accordance with this, small-angle X-ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closure.
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 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: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences , Magyar tudósok krt. 2., H-1117 Budapest, Hungary.
authors.forename: Anita
authors.initials: A
authors.lastname: Garamszegi
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: Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics , Gellért tér 4., H-1111 Budapest, Hungary.
authors.forename: Anikó
authors.initials: A
authors.lastname: Lábas
authors.affiliation: Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics , Gellért tér 4., H-1111 Budapest, Hungary.
authors.forename: Julianna
authors.initials: J
authors.lastname: Oláh
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: European Molecular Biology Laboratory , Hamburg Outstation, Notkestrasse 85, 22603 Hamburg, Germany.
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.affiliation: Dipartimento di Bioscienze, Universitá degli Studi di Parma , 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: Macromolecular Crystallography (HZB-MX), Helmholtz-Zentrum Berlin für Materialien und Energie , Albert-Einstein-Strasse 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.
authors.forename: Mária
authors.initials: M
authors.lastname: Vas
mesh_terms: D050539:3-Isopropylmalate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry*; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D031541:Fluorescence Resonance Energy Transfer; D006863:Hydrogen-Ion Concentration; D056004:Molecular Dynamics Simulation; D009154:Mutation; D017434:Protein Structure, Tertiary; D011789:Quantum Theory; D053838:Scattering, Small Angle; D016963:Thermus thermophilus / Q000201:enzymology*; D014965:X-Rays
chemical_list: D001426:Bacterial Proteins; D050539:3-Isopropylmalate Dehydrogenase