The biochemical basis for the therapeutic effects of thiamin in thiamin-responsive MSUD was investigated in intact and disrupted fibroblast cultures from normal subjects and patients with various forms of MSUD. Decarboxylation of [1-14C]KIV by intact cells from thiamin-responsive MSUD 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. BCKA dehydrogenase activity measured in disrupted cells from the thiamin-responsive subject exhibited sigmoidal kinetics in the absence of TPP with an elevated Km value of 7 mM for KIV. 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 Km value of 4 mM for KIV, suggesting a TPP-mediated increase in the affinity of the mutant enzyme for substrate. By contrast, TPP increased only Vmax and was without effect on the apparent Km for KIV of BCKA dehydrogenase from normal subjects, classical MSUD, and a thiamin-non-responsive MSUD variant (Grade 3). Measurement of apparent Km for TPP of the BCKA dehydrogenase showed a 16-fold increase in the constant to 25 microM for the thiamin-responsive mutant compared to normal or classical MSUD enzymes. 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.