title: Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis.
abstract: The three-dimensional structure of the enzyme 3-isopropylmalate dehydrogenase from the bacterium Thermus thermophilus in complex with Mn(2+) , its substrate isopropylmalate and its co-factor product NADH at 2.0 Å resolution features a fully closed conformation of the enzyme. Upon closure of the two domains, the substrate and the co-factor are brought into precise relative orientation and close proximity, with a distance between the C2 atom of the substrate and the C4N atom of the pyridine ring of the co-factor of approximately 3.0 Å. The structure further shows binding of a K(+) ion close to the active site, and provides an explanation for its known activating effect. Hence, this structure is an excellent mimic for the enzymatically competent complex. Using high-level QM/MM calculations, it may be demonstrated that, in the observed arrangement of the reactants, transfer of a hydride from the C2 atom of 3-isopropylmalate to the C4N atom of the pyridine ring of NAD(+) is easily possible, with an activation energy of approximately 15 kcal·mol(-1) . The activation energy increases by approximately 4-6 kcal·mol(-1) when the K(+) ion is omitted from the calculations. In the most plausible scenario, prior to hydride transfer the ε-amino group of Lys185 acts as a general base in the reaction, aiding the deprotonation reaction of 3-isopropylmalate prior to hydride transfer by employing a low-barrier proton shuttle mechanism involving a water molecule.
  
  
  METHODS
  Structural data have been submitted to the Protein Data Bank under accession number 4F7I.
authors.affiliation: Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Anna
authors.initials: A
authors.lastname: Palló
authors.forename: Julianna
authors.initials: J
authors.lastname: Oláh
authors.forename: Eva
authors.initials: E
authors.lastname: Gráczer
authors.forename: Angelo
authors.initials: A
authors.lastname: Merli
authors.forename: Péter
authors.initials: P
authors.lastname: Závodszky
authors.forename: Manfred S
authors.initials: MS
authors.lastname: Weiss
authors.forename: Mária
authors.initials: M
authors.lastname: Vas
mesh_terms: D050539:3-Isopropylmalate Dehydrogenase / Q000737:chemistry*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D008274:Magnesium / Q000737:chemistry; D008293:Malates / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D008293:Malates; D009243:NAD; D008345:Manganese; D050539:3-Isopropylmalate Dehydrogenase; D008274:Magnesium; D011188:Potassium
keywords: QM/MM calculations; X-ray crystallography; general base catalysis; isopropylmalate dehydrogenase; oxidative decarboxylation