正常人和经典型枫糖尿症患者皮肤成纤维细胞中支链2-氧代酸脱氢酶及其组分的活性

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

作者信息

Chuang D T, Niu W L, Cox R P

出版信息

Biochem J. 1981 Oct 15;200(1):59-67. doi: 10.1042/bj2000059.

DOI:10.1042/bj2000059

PMID:6895847

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1163502/

Abstract

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 Vmax. and apparent Km 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 K0.5 values from the Hill plots for MSUD patients were 4-7 mM. 4. 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 Km or K0.5 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.

摘要

对正常人和经典型枫糖尿症（MSUD）患者培养的皮肤成纤维细胞中支链2-氧代酸脱氢酶的活性和动力学进行比较，为该酶缺陷提供了动力学解释。2. 在完整细胞测定中，正常成纤维细胞以3-甲基-2-氧代[1-¹⁴C]丁酸为底物表现出双曲线动力学。来自四名经典型MSUD患者的完整成纤维细胞在0.25至5.0 mM的底物浓度范围内未显示出脱羧作用，且硫胺素（4 mM）无效。3. 通过使用破碎细胞系统有效地重建了多酶复合物的整体反应。正常人在2-氧代酸浓度为0.1至5 mM时再次表现出典型的双曲线动力学。以3-甲基-2-氧代丁酸为底物时，Vmax.和表观Km值分别为0.10±0.02 m单位/毫克蛋白质和0.05 - 0.1 mM。相比之下，经典型MSUD患者表现出S形动力学（希尔系数为2.5），在5 mM底物时活性接近正常值的40 - 60%。MSUD患者希尔图的K0.5值为4 - 7 mM。4. 在破碎的颗粒制剂中测量了多酶复合物的E1（支链2-氧代酸脱羧酶）成分。正常人再次以2-氧代酸表现出双曲线动力学，而MSUD制剂表现出S形动力学，E1的活性严格依赖于底物浓度。正常人和MSUD患者的表观Km或K0.5分别为0.1和1.0 mM。5. 在MSUD制剂中对E2（二氢硫辛酰胺转乙酰酶）和E3（二氢硫辛酰胺脱氢酶）的测量表明它们在正常范围内。6. 上述数据表明经典型MSUD患者的支链2-氧代酸脱氢酶的E1步骤存在缺陷。