Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis

Anna Palló, J. Oláh, Eva Gráczer, Angelo Merli, P. Závodszky, Manfred S. Weiss, M. Vas

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

UNLABELLED: 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.

DATABASE: Structural data have been submitted to the Protein Data Bank under accession number 4F7I.

Original languageEnglish
Pages (from-to)5063-5076
Number of pages14
JournalFEBS Journal
Volume281
Issue number22
DOIs
Publication statusPublished - Nov 1 2014

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Catalysis
NAD
3-Isopropylmalate Dehydrogenase
Oxidoreductases
Hydrides
Ions
Thermus thermophilus
Atoms
Enzymes
Protons
Catalytic Domain
Substrates
Activation energy
Databases
Bacteria
Deprotonation
Water
Conformations
Proteins
Molecules

Keywords

  • general base catalysis
  • isopropylmalate dehydrogenase
  • oxidative decarboxylation
  • QM/MM calculations
  • X-ray crystallography

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis. / Palló, Anna; Oláh, J.; Gráczer, Eva; Merli, Angelo; Závodszky, P.; Weiss, Manfred S.; Vas, M.

In: FEBS Journal, Vol. 281, No. 22, 01.11.2014, p. 5063-5076.

Research output: Contribution to journalArticle

Palló, Anna ; Oláh, J. ; Gráczer, Eva ; Merli, Angelo ; Závodszky, P. ; Weiss, Manfred S. ; Vas, M. / Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis. In: FEBS Journal. 2014 ; Vol. 281, No. 22. pp. 5063-5076.
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abstract = "UNLABELLED: 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 {\AA} 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 {\AA}. 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.",
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AU - Vas, M.

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N2 - UNLABELLED: 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.DATABASE: Structural data have been submitted to the Protein Data Bank under accession number 4F7I.

AB - UNLABELLED: 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.DATABASE: Structural data have been submitted to the Protein Data Bank under accession number 4F7I.

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