GLUTARYL-COA DEHYDROGENASE AND NEUROLOGIC DISEASE

黄蛋白、戊二酰辅酶A脱氢酶（GCD）的缺陷会导致I型戊二酸尿症（GA 1），这是一种常染色体隐性遗传的神经代谢疾病。GCD通过沿着酰基辅酶A脱氢酶的常见机制催化戊二酰辅酶A的α、β脱氢。GCD还催化酶结合的中间体戊烯二酰辅酶A脱羧为巴豆酰辅酶A和CO2。戊烯二酰辅酶A的脱羧需要黄素脱氢酶的氧化和巴豆酰辅酶A阴离子（-CH 2 =-CH=-CH=-COSCoA）的质子化。在GCD缺陷的患者中，GA 1患者的神经系统症状的发作通常在生命早期的病毒注射之后。已经鉴定了超过50种错义突变，其可能影响戊二酰辅酶A的氧化和脱羧。这些突变还可能影响四聚体的组装或稳定性、黄素脱氢酶的氧化还原电位或黄素脱氢酶通过电子转移黄素蛋白（ETF）的再氧化。拟议的研究有以下具体目标。[1]将表达许多突变等位基因，并通过动力学和氧化还原方法表征缺陷蛋白，以获得酶缺陷的基础。[2]我们将研究偶联戊二酰辅酶A氧化与脱羧/质子化的酶结合的中间体，戊烯二酰辅酶A，使用定点突变假设解耦这些步骤的催化。将确定具有结合的戊二酰辅酶A类似物和具有反应中间体戊烯二酰辅酶A的GCD的晶体结构，以提供对脱羧反应的了解。[3]我们将直接研究戊烯二酰辅酶A的脱羧作用，并确定特定氨基酸在反应中的作用。还将确定FAD辅基的核糖基侧链的2 '-羟基在脱羧/质子化中间体的稳定中的参与。

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.