Thiamin-responsive maple-syrup-urine disease: Decreased affinity of the mutant branched-chain α-keto acid dehydrogenase for α-ketoisovalerate and thiamin pyrophosphate

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Abstract

The biochemical basis for the therapeutic effects of thiamin in thiamin-responsive maple-syrup-urine disease (MSUD) was investigated in intact and disrupted fibroblast cultures from normals and patients wth various forms of MSUD. Decarboxylation of α-keto[1-14C]isovalerate (KIV) by intact cells from a thiamin-responsive MSUD patient was at 30-40% of the normal rate with or without thiamin in the incubation medium. Under similar conditions, intact classical MSUD fibroblasts failed to decarboxylate KIV. Branched-chain α-keto acid (BCKA) dehydrogenase activity measured in disrupted cells from the thiamin-responsive subject showed sigmoidal kinetics in the absence of thiamin pyrophosphate (TPP), with an increased concentration of substrate needed for half-maximal velocity (K(0.5) for KIV=7 mM vs. 0.05 mM in normal cells). When assayed with 0.2 mM TPP present, the mutant enzyme showed a shift in kinetics to near Michaelis-Menten type as observed with the normal BCKA dehydrogenase and a lower K(0.5) value of 4 mM for KIV, suggesting a TPP-mediated increase in the mutant enzyme's affinity for substrate. By contrast, TPP increased only the V(max) and was without effect on the apparent K(m) for KIV of the BCKA dehydrogenase from cells of normals and patients with classical MSUD and variant thiamin-nonresponsive MSUD (grade 3). Measurement of the apparent K(m) for TPP of the BCKA dehydrogenase from thiamin-responsive mutant MSUD cells showed a 16-fold increase in the constant to 25 μM compared to enzymes from normal or classical MSUD cells. These findings demonstrate that the primary defect in the thiamin-responsive MSUD patient is a reduced affinity of the mutant BCKA dehydrogenase for TPP that results in impaired oxidative decarboxylation of BCKA.

Original languageEnglish (US)
Pages (from-to)3300-3304
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume79
Issue number10 I
DOIs
StatePublished - Jan 1 1982

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