Activities of branched-chain 2-oxo acid dehydrogenase and its components in skin fibroblasts from normal and classical maple-syrup-urine-disease subjects

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Comparisons of the activity and kinetics of the branched-chain 2-oxo acid dehydrogenase in cultured skin fibroblasts from normal and classical maple-syrup-urine disease (MSUD) subjects provide a kinetic explanation for the enzyme defect. 2. In the intact cell assays, normal fibroblasts demonstrated hyperbolic kinetics with 3-methyl-2-oxo[1-14C]butyrate as a substrate. Intact fibroblasts from four classical MSUD patients showed no decarboxylation over a substrate concentration range of 0.25 to 5.0 mM, and thiamin (4 mM) was without effect. 3. The overall reaction of the multienzyme complex was efficiently reconstituted by using a disrupted-cell system. Normals again showed typical hyperbolic kinetics at the 2-oxo acid concentrations of 0.1 to 5 mM. The V(max), and apparent K(m) values were 0.10 ± 0.02 m-unit/mg of protein and 0.05-0.1 mM respectively, with 3-methyl-2-oxobutyrate. In contrast, classical MSUD patients exhibited sigmoidal kinetics (Hill coefficient, 2.5) with activity approaching 40-60% of the normal value at 5 mM substrate. The K(0.5) values from the Hill plots for MSUD patients were 4-7 mM. The E1 (branched-chain 2-oxo acid decarboxylase) component of the multienzyme complex was measured in disrupted-particulate preparations. Normals again showed hyperbolic kinetics with the 2-oxo acid, whereas MSUD preparations exhibited sigmoidal kinetics with the activity of E1 strictly dependent on substrate concentration. Apparent K(m) or K(0.5) were 0.1 and 1.0 mM for normal and MSUD subjects respectively. 5. Measurements of E2 (dihydrolipoyl transacylase) and E3 (dihydrolipoyl dehydrogenase) in MSUD preparations showed them to be in the normal range. 6. The above data suggest a defect in the E1 step of branched-chain 2-oxo acid dehydrogenase in classical MSUD patients.

Original languageEnglish (US)
Pages (from-to)59-67
Number of pages9
JournalBiochemical Journal
Issue number1
StatePublished - Jan 1 1981


ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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