THIAMIN-RESPONSIVE MAPLE-SYRUP-URINE DISEASE - DECREASED AFFINITY OF THE MUTANT BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE FOR ALPHA-KETOISOVALERATE AND THIAMIN PYROPHOSPHATE
THIAMIN-RESPONSIVE MAPLE-SYRUP-URINE DISEASE - DECREASED AFFINITY OF THE MUTANT BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE FOR ALPHA-KETOISOVALERATE AND THIAMIN PYROPHOSPHATE
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DOI：
10.1073/pnas.79.10.3300
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发表时间：
1982-01-01
期刊：
影响因子：
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通讯作者：
COX, RP
中科院分区：
文献类型：
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作者：
CHUANG, DT;KU, LS;COX, RP
在正常人和患有各种形式的 MSUD 患者的完整和破坏的成纤维细胞培养物中，研究了硫胺素对硫胺素反应性枫糖浆尿病 (MSUD) 的治疗作用的生化基础。来自硫胺素响应性MSUD患者的完整细胞对α-酮[1-14C]异戊酸(KIV)的脱羧率为正常速率的30-40％，无论培养介质中是否含有硫胺素。在类似条件下，完整的经典 MSUD 成纤维细胞未能使 KIV 脱羧。在来自硫胺素反应性受试者的破碎细胞中测量的支链α-酮酸(BCKA)脱氢酶活性在不存在焦磷酸硫胺素(TPP)的情况下显示出S形动力学，其中半最大速度所需的底物浓度增加(KIV的K0.5=7mM对比正常细胞中的0.05mM)。当在存在 0.2 mM TPP 的情况下进行分析时，突变酶显示出与正常 BCKA 脱氢酶观察到的接近 Michaelis-Menten 类型的动力学转变，并且 KIV 的 K0.5 值较低为 4 mM，表明 TPP 介导的突变酶对底物的亲和力增加。相比之下，TPP 仅增加 Vmax，并且对来自正常细胞和患有经典 MSUD 和变异硫胺素无反应 MSUD 的患者细胞的 BCKA 脱氢酶 KIV 的表观 Km 没有影响（3 级）。对来自硫胺素应答突变体MSUD细胞的BCKA脱氢酶的TPP的表观Km的测量显示，与来自正常或经典MSUD细胞的酶相比，常数增加了16倍至25μM。硫胺素反应性 MSUD 患者的主要缺陷是突变 BCKA 脱氢酶对 TPP 的亲和力降低，导致 BCKA 氧化脱羧受损。
The biochemical basis for the therapeutic effects of thiamin in thiamine-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 thiamine-responsive MSUD patient was at 30-40% of the normal rate with or without thiamine 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 thiamine-responsive subject showed sigmoidal kinetics in the absence of thiamine 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 a shift in kinetics to near Michaelis-Menten type as observed with the normal BCKA dehydrogenase and 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 thiamine-nonresponsive MSUD (grade 3). Measurement of the apparent Km for TPP of the BCKA dehydrogenase from thiamine-responsive mutant MSUD cells showed a 16-fold increase in the constant to 25 .mu.M compared to enzymes from normal or classical MSUD cells. The primary defect in the thiamine-responsive MSUD patient is a reduced affinity of the mutant BCKA dehydrogenase for TPP that results in impaired oxidative decarboxylation of BCKA.