GLUTARYL-COA DEHYDROGENASE AND NEUROLOGIC DISEASE

戊二酰辅酶a脱氢酶（GCD）黄蛋白缺陷导致戊二酸尿I型（GA1），这是一种常染色体隐性遗传的神经代谢疾病。GCD通过与酰基辅酶a脱氢酶相同的机制催化戊二酰辅酶a脱氢。GCD还催化酶结合的中间体谷氨酰基辅酶a脱羧为巴豆酰辅酶a和CO2。谷丙酰辅酶a的脱羧需要脱氢酶黄素的氧化和所提出的谷丙酰辅酶a阴离子的质子化（-CH2=-CH=-CH=-COSCoA）。在GCD缺陷患者中，GA1患者的神经系统症状通常在生命早期进行病毒注射后出现。超过50种错义突变可能影响戊二酰辅酶a的氧化和脱羧。这些突变也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。提出的研究有以下具体目的。许多突变等位基因将被表达，缺陷蛋白将通过动力学和氧化还原方法表征，以获得酶缺陷的基础。我们将研究谷氨酰基辅酶a氧化与酶结合中间体谷氨酰基辅酶a脱羧/质子化的偶联，使用假设的位点定向突变来解耦催化过程中的这些步骤。结合戊二酰辅酶a类似物和反应中间体谷氨酰辅酶a的GCD的晶体结构将被确定，为脱羧反应提供洞察。我们将直接研究谷氨酰基辅酶a的脱羧反应，并确定特定氨基酸在反应中的作用。还将确定FAD假体基利比特侧链的2'-羟基在脱羧/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.