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.

Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis / Palló, Anna; Oláh, Julianna; Gráczer, Eva; Merli, Angelo; Závodszky, Péter; Weiss, Manfred S; Vas, Mária. - In: THE FEBS JOURNAL. - ISSN 1742-464X. - 281:22(2014), pp. 5063-5076. [10.1111/febs.13044]

Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis

MERLI, Angelo;
2014

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.
Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis / Palló, Anna; Oláh, Julianna; Gráczer, Eva; Merli, Angelo; Závodszky, Péter; Weiss, Manfred S; Vas, Mária. - In: THE FEBS JOURNAL. - ISSN 1742-464X. - 281:22(2014), pp. 5063-5076. [10.1111/febs.13044]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2787452
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