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 with various forms of MSUD. Decarboxylation of alpha-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 alpha-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 (K0.5 for KIV = 7 mM vs. 0.05 mM in normal cells). When assayed with 0.2 mM TPP present, the mutant
enzyme showed (i) a shift in kinetics to near Michaelis-Menten type as observed with the normal BCKA
dehydrogenase and (ii) a lower K0.5 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 Vmax and was without effect on the apparent Km for KIV of the BCKA
dehydrogenase from cells of normals and patients with classical MSUD and variant
thiamin-responsive MSUD (grade 3). Measurement of the apparent Km for TPP of the BCKA
dehydrogenase from
thiamin-responsive mutant MSUd cells showed a 16-fold increase in the constant to 25 microM 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.