Structural and biochemical characterization of human mitochondrial branched-chain α-ketoacid dehydrogenase phosphatase

Richard M Wynn, Jun Li, Chad A Brautigam, Jacinta L. Chuang, David T Chuang

Research output: Contribution to journalArticle

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Abstract

The branched-chain α-ketoacid dehydrogenase phosphatase (BDP) component of the human branched-chain α-ketoacid dehydrogenase complex (BCKDC) has been expressed in Escherichia coli and purified in the soluble form. The monomeric BDP shows a strict dependence on Mn 2+ ions for phosphatase activity, whereas Mg 2+ and Ca 2+ ions do not support catalysis. Metal binding constants for BDP, determined by competition isothermal titration calorimetry, are 2.4 nM and 10μM for Mn 2+ and Mg 2+ ions, respectively. Using the phosphorylated decarboxylase component (p-E1b) of BCKDC as a substrate, BDP shows a specific activity of 68 nmol/min/mg. The Ca 2+-independent binding of BDP to the 24-meric transacylase (dihydrolipoyl transacylase; E2b) core of BCKDC results in a 3-fold increase in the dephosphorylation rate of p-E1b. However, the lipoyl prosthetic group on E2b is not essential for BDP binding or E2b-stimulated phosphatase activity. Acidic residues in the C-terminal linker of the E2b lipoyl domain are essential for the interaction between BDP and E2b. The BDP structure was determined by x-ray crystallography to 2.4 Å resolution. The BDP structure is dominated by a central β-sandwich. There are two protrusions forming a narrow cleft ∼10 Å wide, which constitutes the active site. The carboxylate moieties of acidic residues Asp-109, Asp-207, Asp-298, and Asp-337 in the active-site cleft participate in binding two metal ions. Substitutions of these residues with alanine nullify BDP phosphatase activity. Alteration of the nearby Arg-104 increases the K m for p-E1b peptide by 60-fold, suggesting that this residue is critical for the recognition of the native p-E1b protein.

Original languageEnglish (US)
Pages (from-to)9178-9192
Number of pages15
JournalJournal of Biological Chemistry
Volume287
Issue number12
DOIs
StatePublished - Mar 16 2012

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3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
Phosphoric Monoester Hydrolases
Ions
Catalytic Domain
Metals
Calorimetry
Crystallography
Carboxy-Lyases
Prosthetics
Catalysis
Titration

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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Structural and biochemical characterization of human mitochondrial branched-chain α-ketoacid dehydrogenase phosphatase. / Wynn, Richard M; Li, Jun; Brautigam, Chad A; Chuang, Jacinta L.; Chuang, David T.

In: Journal of Biological Chemistry, Vol. 287, No. 12, 16.03.2012, p. 9178-9192.

Research output: Contribution to journalArticle

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abstract = "The branched-chain α-ketoacid dehydrogenase phosphatase (BDP) component of the human branched-chain α-ketoacid dehydrogenase complex (BCKDC) has been expressed in Escherichia coli and purified in the soluble form. The monomeric BDP shows a strict dependence on Mn 2+ ions for phosphatase activity, whereas Mg 2+ and Ca 2+ ions do not support catalysis. Metal binding constants for BDP, determined by competition isothermal titration calorimetry, are 2.4 nM and 10μM for Mn 2+ and Mg 2+ ions, respectively. Using the phosphorylated decarboxylase component (p-E1b) of BCKDC as a substrate, BDP shows a specific activity of 68 nmol/min/mg. The Ca 2+-independent binding of BDP to the 24-meric transacylase (dihydrolipoyl transacylase; E2b) core of BCKDC results in a 3-fold increase in the dephosphorylation rate of p-E1b. However, the lipoyl prosthetic group on E2b is not essential for BDP binding or E2b-stimulated phosphatase activity. Acidic residues in the C-terminal linker of the E2b lipoyl domain are essential for the interaction between BDP and E2b. The BDP structure was determined by x-ray crystallography to 2.4 {\AA} resolution. The BDP structure is dominated by a central β-sandwich. There are two protrusions forming a narrow cleft ∼10 {\AA} wide, which constitutes the active site. The carboxylate moieties of acidic residues Asp-109, Asp-207, Asp-298, and Asp-337 in the active-site cleft participate in binding two metal ions. Substitutions of these residues with alanine nullify BDP phosphatase activity. Alteration of the nearby Arg-104 increases the K m for p-E1b peptide by 60-fold, suggesting that this residue is critical for the recognition of the native p-E1b protein.",
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