人類二氫硫辛酸去氫酶之構造與功能的研究

哺乳類動物的酮酸去氫酉每複合體（α－keto acid dehydrogenase complex） 包括：丙酮酸去氫酉每複合體（pyruvate dehydrogenase complex, PDC）酮戊醯酸去氫酉每複合體（α-ketoglutarate dehydrogenase complex, α-KGDC）及側鏈酮酸去氫酉每複合體（branched chain α-keto acid dehydrogenase complexes, BCKADC）等三種。此類酵素複合體能將其受質經氧化脫羧產生 acetyl-CoA、CO2及 NADH。　　酮酸去氫酉每複合體位於粒線內，它是由酮酸去氫酉每（α－Keto acid dehydrogenase, E1）、二氫硫辛酸醯基轉移酉每（dihydrolipoamide acetyltransferase, E2）及二氫硫辛酸去氫酉每（dihydrolipoamide dehydrogenase, E2）等三種酵素依特定比例組成複合體，進行酵素之催化反應。　　上述三種酵素中，Ｅ３是酮酸去氫酉每複合體的共同組成部分，所以當Ｅ３酵素活性發生缺乏時，三種酮酸去氫酉每複合體的活性均會受到影響。因此Ｅ３在能量代謝上扮演很重要的角色，缺乏Ｅ３的酵素活性會造成能量代謝嚴重的障礙。　　因為Ｅ３是能量代謝上重要的酵素，所以本實驗的研究目的是研究Ｅ３的反應機制，尤其是Ｅ３反應時電子傳遞的路徑。在方法學上，本實驗是利用定點突變（SDMPCR）製造Ｅ３的突變蛋白質（K54E;SK-KS），並利用分子量測定、酵素動力學、螢光分析、FAD含量測定等方法探討酵素構造和功能間的關係。　　分子量測定（molecular sieving）發現三種蛋白質的分子量大致相同大約為100kDa；換言之，突變本身不會影響酵素形成同質雙體（homodimer）的性質。酵素動力學研究（enzyme kinetic analysis）的結果顯示Ｅ３及SK-KS的反應機制是屬於雙受質的乒乓反應（ping pong mechanism）。蛋白質相對的FAD含量比Ｅ３：SK-KS：K54E為100：82：90。蛋白質相對的比活性（specific activity）比 E3：SK-KS： K54E為100：46：1.7。螢光分析（fluorescence analysis）的結果顯示Ｅ３的ＦＡＤ會被二氫硫辛酸去氫酉每還原成FADH，然後ＦＡＤＨ再進一步的將ＮＡＤ＋還原產生NADH；此一步驟在突變種明顯的比野生種慢。　　綜合上述結果，推測Ｅ３cDNA上的第54號胺基酸（Lysine）參與電子傳遞，且可能是酵素活性中心的成員之一，此胺基酸對於酵素的催化活性有關鍵性的影響。

Mammalian α-keto acid dehydrogenase complexes includes pyruvate dehydrogenase comples (PDC),α-ketoglutarate dehydrogenase (αKGDC),branched chain α-keto acid dehydrogenase complexes(BCKADC). These enzyme complexes catalyze the oxidative decarhboxylation of α-keto acid with the formation of acyl-CoA, CO2 and NADH. The α-keto acid dehydrogenase complexes are found in the mitochondria. Each enzyme complex is composed of three catalytic components: α-keto acid dehydogenase (E1),dihydrolipoamide acyltransferase (E2) and dihydrolipoaimide dehydrogenase (E3). E1 and E2 show substrates specificity and E3 is the common component for all α-keto acid dehydrogenase complexes .In case of E3 deficiency , it will affect all α-keto acid dehydrogenase complexes.　　Since E3 plays an important role in the energy metabolism. The approach of this study is characterizing the electron transferring pathway of E3 by using site-directed mutagenesis (SDM).　　To investigate the role of lysine-54 of human dihydrolipoamide dehydrogenase (E3) in the formmation of intermediate during electrons transfer two mutant human E3s, S53K54-K53S54 (SK-KS) and K54E, were overexpressed, purified and characterized. Molecular sieving analysis showed that each proteins (E3, SK-KS, K54E )was dimer with the molecular weight approximately to 100 kDa. The results of kinetic analysis show the E3 and ES-KS following two substrates'' ping pong mechanism, The relative FAD content of the enzymes is E3:SK-KS:K54E=100:82:90 .The relative specifc activity of the enzymes is E3:SK-KS:K54E=100:46:1.7.The fluorescence analysis show that E3'' FAD can be reduced by DHL and then formed FADH.SK-KS mutant''s FADH reoxidation is significantly slowly than the wild type E3 and K54E mutant''s FAD is extremely could not be reduced by DHL. The FADH will be oxidized by NAD+with the NADH formation, At this stage, the mutant proteins (SK-KS and K54E) are significantly slowly than the wild type E3 .The data showed that the number 54th amino acid (K54) of E3 cDNA maybe participate in the enzyme''s active center and specifically significantly corresponding for enzyme''s activity.