雙羥擬脂醯胺去氫(dihydrolipoamide dehydrogenase; E3)是屬於嘌呤核雙硫氧化還原家族之一。此族群主要特徵是具有活性雙硫基(active disulfide)、活性鹼基(active base)和FAD，其活性中心可在電子接受者(NAD+或NADP+)與雙硫基受質(dithiol substrate)之間傳遞電子；E3也是哺乳類的酮酸去氫複合體包括丙酮酸去氫複合體(pyruvate dehydrogenase complex, PDC)、α-酮戊二酸去氫複合體(α-ketoglutarate dehydrogenase complex, KGDC)及側鏈酮酸去氫複合體(branched-chain α-keto acid dehydrogenase complex, BCKADC)等三種複合體的共同組成成份，所以當E3發生缺乏或有缺陷時，三種酮酸去氫複合體的活性均會受到影響，因此E3在能量代謝上佔有舉足輕重的地位。所以本實驗研究的目的即是探討E3：E192在E3反應機制及電子傳遞路徑上扮演的角色。其研究方法首先利用定點突變方法創造出E192Q這個突變蛋白質，隨後利用分子量測定、FAD含量測定、酵素動力學分析、光譜分析及氧化還原電位測定等方法，來探討酵素結構與功能間的關係。 經由分子量測定發現突變蛋白質與正常蛋白質其分子量大致相同，約為100kDa；換言之突變並不會影響其同質雙體(homodimer)的結構。酵素相對的FAD含量比E3：E192Q為100：95。而在酵素動力學分析中E192Q之代謝迴轉速率(Kcat)約只為正常E3的10%。令人驚奇的是E192Q突變蛋白正反應的反應機制從正常E3的乒乓反應(ping pong mechanism)變成序列機制(sequential mechanism)。進一步在螢光光譜分析中發現E192Q突變蛋白從DHL傳遞電子至FAD形成FADH，再進一步的將NAD+還原產生NADH之反應途徑幾乎完全被阻斷，隨後的紫外光/可見光光譜分析則發現E192Q突變蛋白在530nm雙硫基與FAD間的電價轉移階段其電子傳遞緩慢而近乎停滯；而在氧化還原電位中點分析中，E192Q突變蛋白的FAD還原電位中點和E3相比則發現有升高的現象產生。 由以上結果推論E192Q的代謝迴轉率的大幅下降可能是因為突變致使FAD無法被順利還原成FADH進而使電子傳遞路徑的受阻所導致。所以推測E192參與電子傳遞，可能是E3活性中心的成員之一，其對於活化中心的穩定和酵素功能的維持具有關建性的影響，在E 3的反應機制中確實扮演一個非常重要的角色。 Dihydrolipoamide dehydrogenase(E3) belongs to the enzyme family of pyridine nucleotide-disulfide oxidoreductases and catalyzes the transfer of electrons from dihydrolipoamide to FAD cofactor , then to the NAD＋. E3is also the common component of α-ketoacid dehydrogenase complexes including pyruvate dehydrogenase (PDC) , branched-chain α-ketoacid dehydrogenas complexes and α-ketoglutarate dehydrogenas complexes. A deficiency in E3 leads to the deficiency of all these threeα-ketoacid dehydrogenase complexes. E3 plays an important role in the energy metabolism indeed. This study is to characterize the functional significant of E-192 in the reaction mechanism and to focused on the role of E-192 in electron transferring pathway. The approach in this study was using site-directed mutagenesis(SDM) to create the E192Q mutant protein, and the mutant protein was subjected into the following analysis which including molecular sieving analysis, relative FAD content assay , enzyme kinetics assay , spectrophotometer analysis and midpoint reduction potential analysis. Molecular sieving analysis showed that the E192Q mutant protein was homodimer with the molecular-weight approximately to 102 kDa, as well as wild-type E3. The relative FAD content of these enzymes are E3：E192Q＝100：95.The Kcat of E192Q is reduced to 5%, while compared to wild-type E3. It is interesting that the reaction mechanism of E192Q is sequential mechanism while E3 is the ping pong mechanism in forward reaction. The fluorescence assay reveals that the FAD can be reduced by DHL with the formation of FADH in E3. The FADH can be further oxidized by NAD＋with the NADH formation. E192Q almost loss the ability of electrons transferring between FAD to NAD＋when compared to E3. In addition, the UV/VIS spectrophotometer analysis of E192Q indicates that the electron transferring from active disulfide to FAD may be blocked. Further, the midpoint potential of FAD is also increased. In conclusion, the E192 maybe involve in the active center of E3 and specifically corresponding to the reduction of FAD in the E3.reaction mechanism.
