Structural determinants for branched-chain aminotransferase isozyme-specific inhibition by the anticonvulsant drug gabapentin

Masaru Goto, Ikuko Miyahara, Ken Hirotsu, Myra Conway, Neela Yennawar, Mohammad M. Islam, Susan M. Hutson

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

This study presents the first three-dimensional structures of human cytosolic branched-chain aminotransferase (hBCATc) isozyme complexed with the neuroactive drug gabapentin, the hBCATc Michaelis complex with the substrate analog, 4-methylvalerate, and the mitochondrial isozyme (hBCATm) complexed with gabapentin. The branched-chain aminotransferases (BCAT) reversibly catalyze transamination of the essential branched-chain amino acids (leucine, isoleucine, valine) to α-ketoglutarate to form the respective branched-chain α-keto acids and glutamate. The cytosolic isozyme is the predominant BCAT found in the nervous system, and only hBCATc is inhibited by gabapentin. Pre-steady state kinetics show that 1.3 mM gabapentin can completely inhibit the binding of leucine to reduced hBCATc, whereas 65.4 mM gabapentin is required to inhibit leucine binding to hBCATm. Structural analysis shows that the bulky gabapentin is enclosed in the active-site cavity by the shift of a flexible loop that enlarges the active-site cavity. The specificity of gabapentin for the cytosolic isozyme is ascribed at least in part to the location of the interdomain loop and the relative orientation between the small and large domain which is different from these relationships in the mitochondrial isozyme. Both isozymes contain a CXXC center and form a disulfide bond under oxidizing conditions. The structure of reduced hBCATc was obtained by soaking the oxidized hBCATc crystals with dithiothreitol. The close similarity in active-site structures between cytosolic enzyme complexes in the oxidized and reduced states is consistent with the small effect of oxidation on pre-steady state kinetics of the hBCATc first half-reaction. However, these kinetic data do not explain the inactivation of hBCATm by oxidation of the CXXC center. The structural data suggest that there is a larger effect of oxidation on the interdomain loop and residues surrounding the CXXC center in hBCATm than in hBCATc.

Original languageEnglish (US)
Pages (from-to)37246-37256
Number of pages11
JournalJournal of Biological Chemistry
Volume280
Issue number44
DOIs
StatePublished - Nov 4 2005

Fingerprint

Anticonvulsants
Isoenzymes
Leucine
Catalytic Domain
Oxidation
Kinetics
Keto Acids
Branched Chain Amino Acids
Essential Amino Acids
Isoleucine
Dithiothreitol
Valine
Neurology
gabapentin
branched-chain-amino-acid transaminase
Structural analysis
Disulfides
Nervous System
Glutamic Acid
Crystals

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Goto, Masaru ; Miyahara, Ikuko ; Hirotsu, Ken ; Conway, Myra ; Yennawar, Neela ; Islam, Mohammad M. ; Hutson, Susan M. / Structural determinants for branched-chain aminotransferase isozyme-specific inhibition by the anticonvulsant drug gabapentin. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 44. pp. 37246-37256.
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Structural determinants for branched-chain aminotransferase isozyme-specific inhibition by the anticonvulsant drug gabapentin. / Goto, Masaru; Miyahara, Ikuko; Hirotsu, Ken; Conway, Myra; Yennawar, Neela; Islam, Mohammad M.; Hutson, Susan M.

In: Journal of Biological Chemistry, Vol. 280, No. 44, 04.11.2005, p. 37246-37256.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Structural determinants for branched-chain aminotransferase isozyme-specific inhibition by the anticonvulsant drug gabapentin

AU - Goto, Masaru

AU - Miyahara, Ikuko

AU - Hirotsu, Ken

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AU - Yennawar, Neela

AU - Islam, Mohammad M.

AU - Hutson, Susan M.

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N2 - This study presents the first three-dimensional structures of human cytosolic branched-chain aminotransferase (hBCATc) isozyme complexed with the neuroactive drug gabapentin, the hBCATc Michaelis complex with the substrate analog, 4-methylvalerate, and the mitochondrial isozyme (hBCATm) complexed with gabapentin. The branched-chain aminotransferases (BCAT) reversibly catalyze transamination of the essential branched-chain amino acids (leucine, isoleucine, valine) to α-ketoglutarate to form the respective branched-chain α-keto acids and glutamate. The cytosolic isozyme is the predominant BCAT found in the nervous system, and only hBCATc is inhibited by gabapentin. Pre-steady state kinetics show that 1.3 mM gabapentin can completely inhibit the binding of leucine to reduced hBCATc, whereas 65.4 mM gabapentin is required to inhibit leucine binding to hBCATm. Structural analysis shows that the bulky gabapentin is enclosed in the active-site cavity by the shift of a flexible loop that enlarges the active-site cavity. The specificity of gabapentin for the cytosolic isozyme is ascribed at least in part to the location of the interdomain loop and the relative orientation between the small and large domain which is different from these relationships in the mitochondrial isozyme. Both isozymes contain a CXXC center and form a disulfide bond under oxidizing conditions. The structure of reduced hBCATc was obtained by soaking the oxidized hBCATc crystals with dithiothreitol. The close similarity in active-site structures between cytosolic enzyme complexes in the oxidized and reduced states is consistent with the small effect of oxidation on pre-steady state kinetics of the hBCATc first half-reaction. However, these kinetic data do not explain the inactivation of hBCATm by oxidation of the CXXC center. The structural data suggest that there is a larger effect of oxidation on the interdomain loop and residues surrounding the CXXC center in hBCATm than in hBCATc.

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