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Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis
Author(s) -
Palló Anna,
Oláh Julianna,
Gráczer Éva,
Merli Angelo,
Závodszky Péter,
Weiss Manfred S.,
Vas Mária
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13044
Subject(s) - thermus thermophilus , hydride , chemistry , active site , deprotonation , stereochemistry , substrate (aquarium) , dehydrogenase , crystallography , ring (chemistry) , nad+ kinase , catalysis , ion , enzyme , hydrogen , biology , biochemistry , organic chemistry , ecology , escherichia coli , gene
The three‐dimensional structure of the enzyme 3‐isopropylmalate dehydrogenase from the bacterium T hermus 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. Database Structural data have been submitted to the Protein Data Bank under accession number 4F7I .