GLUTARYL-COA DEHYDROGENASE AND NEUROLOGIC DISEASE

黄素蛋白戊二酰辅酶A脱氢酶(GCD)的缺陷会导致戊二酸尿I型(GA1)，这是一种常染色体隐性遗传性神经代谢紊乱。GCD催化戊二酰辅酶A的α，β脱氢反应的机理与酰基辅酶A脱氢酶一样。GCD还催化酶结合的中间体谷氨酰辅酶A脱羧基为巴豆酰辅酶A和二氧化碳。谷氨酰辅酶A的脱羧基需要脱氢酶黄素的氧化和建议的巴豆酰辅酶A阴离子(-CH2=-CH=-CH=-COSCoA)的质子化。在GCD缺陷的患者中，GA1患者的神经症状通常是在生命早期注射病毒后出现的。已鉴定出50多个错义突变，可能影响戊二酰辅酶A的氧化和脱羧化。这些突变还可能影响四聚体的组装或稳定性、脱氢酶黄素的氧化还原电位或电子转移黄素蛋白(ETF)对脱氢酶黄素的再氧化。建议的研究有以下具体目标。[1]将表达一些突变的等位基因，并通过动力学和氧化还原方法对缺陷蛋白进行表征，以获得酶缺陷的基础。[2]我们将使用定点突变来研究酶结合中间体谷氨酰辅酶A的脱羧基/质子化与戊二酰辅酶A氧化的偶联，假设这些步骤在催化中解偶联。GCD与结合的戊二酰辅酶A类似物和反应中间体谷氨酰辅酶A的晶体结构将被确定，以提供对脱羧基反应的洞察。[3]我们将直接研究谷氨酰辅酶A的脱羧基反应，并确定特定氨基酸在反应中的作用。还将确定FAD假体基团核糖基侧链的2‘-羟基在脱羧化/质子化中间体稳定中的作用。

Defects in the flavoprotein, glutaryl-CoA dehydrogenase (GCD), cause glutaric aciduria type I (GA1), an autosomal recessively inherited neurometabolic disorder. GCD catalyzes the alpha, beta dehydrogenation of glutaryl-CoA by a mechanism which is common along acyl-CoA dehydrogenases. GCD also catalyzes the decarboxylation of the enzyme- bound intermediate, glutaconyl-CoA, to crotonyl-CoA and CO2. Decarboxylation of glutaconyl-CoA requires oxidation of the dehydrogenase flavin and protonation of the proposed crotonyl-CoA anion (-CH2=-CH=-CH=-COSCoA). In patients with defects in GCD, the onset of neurological symptoms in GA1 patients usually follows a viral injection early in life. Over 50 missense mutations have been identified that may affect oxidation and decarboxylation of glutaryl-CoA. These mutations may also affect assembly or stability of the tetramer, the oxidation-reduction potential of the dehydrogenase flavin or reoxidation of the dehydrogenase flavin by electron transfer flavoprotein (ETF). The proposed research has the following specific aims. [1] A number of mutant alleles will be expressed and the defective proteins characterized by kinetic and redox methods to access the basis of the enzymatic defects. [2] We will investigate the coupling of glutaryl-CoA oxidation with decarboxylation/protonation of the enzyme bound intermediate, glutaconyl-CoA, using site directed mutations hypothesized to uncouple these steps in catalysis. The crystal structure of GCD with a bound glutaryl-CoA analog and with the reaction intermediate, glutaconyl-CoA, will be determined to provide insight into the decarboxylation reaction. [3] We will investigate the decarboxylation of glutaconyl-CoA directly and define the roles of specific amino acids in the reaction. The participation of the 2'-hydroxyl of the ribityl side chain of the FAD prosthetic group in stabilization of decarboxylation/protonation intermediates will also be determined.