人類二氫硫辛醯胺去氫酶構造與功能關係之研究

Study of Structure and function Relationship of Human Dihydrolipoamide Dehydrogenase

10.6834/CSMU.2007.00116

王怡鈞

二氫硫辛醯胺去氫酶酵素動力學 ； Dihydrolipoamide Dehydrogenase ； enzyme kinetics

中山醫學大學醫學研究所學位論文

2007年

博士

陳凌雲;劉德中

繁體中文

二氫硫辛醯胺去氫酶 (Dihydrolipoamide Dehydrogenase) 或稱為E3，是酮酸去氫酶複合體中共同的一個次單元，屬於pyridine nucleotide-disulfide oxidoreductases酵素家族中的一個成員，其作用是將電子在NAD+或NADP+與dithiol受質間做轉移。在細胞中有不同的酮酸去氫酶複合體，包括pyruvate dehydrogenase complex (PDC)、α-ketoglutarate dehydrogenase complex (α-KGDC)，與branched-chain α-keto acid dehydrogenase complexes (BCKADC) 等三種。酮酸去氫酶複合體是由酮酸去氫酶(α-keto acid dehydrogenase; E1)、二氫硫辛醯胺烷醯基轉移酶 (dihydrolipoamide acyltransferase; E2) 及二氫硫辛醯胺去氫酶 (dihydrolipoamide dehydrogenase; E3) 等三種酵素所共同組成。酮酸去氫酶複合體中，E1與E2具受質專一性，而E3則是所有複合體所共有。因此，當E3功能異常時，會同時影響到這些酮酸去氫酶複合體的活性。 為了研究E3的反應機制，我們選擇了17種真核生物的E3序列與人類E3序列進行多重序列比對，從比對的結果與人類E3結構的觀察，選擇了四個胺基酸T148、R281、N286與D320進行點突變，共有7個突變種E3分別是T148G、T148S、R281N、R281K、N286D、N286Q與D320N被建構與表答。分析結果顯示，T148G、T148S、R281N、R281K、N286D、N286Q與D320N之比活性為正常E3的76.34%、88.62%、12.50%、11.93%、30.84%、24.57%與48.60%；在FAD含量上則分別是正常E3的71%、92%、96%、93%、96%、99%與82%。分子量分析顯示這些突變種E3都會形成homodimer。酵素動力學的分析顯示，正、逆反應的受質迴轉率 (Kcat) 除了T148的突變種E3沒什麼改變外，其餘的突變種E3都顯著下降。我們的結果顯示除了T148對於酵素的催化功能沒有重大影響外，其餘突變點的胺基酸對於E3的催化功能，均扮演重要角色。

Human dihydrolipoamide dehydrogenase (hE3) is a common component of α-ketoacid dehydrogenase complexes. E3 belongs to the enzyme of pyridine nucleotide-disulfide oxidoreductases. These enzymes have an active disulfide, an active base, and FAD to transfer electrons between the electron acceptor (NAD+ or NADP+) and dithiol substrate in the active site. Mutations to this homodimeric protein cause E3 deficiency and always fatal. To investigate its reaction mechanism, we first performed multiple sequence alignment with other seventeen eukaryotic E3s. According to hE3 structure and the result of multiple sequence alignment, four amino acids T148, R281, N286, and D320 were selected and subjected into mutagenesis. Seven recombinant mutant E3, T148G, T148S, R281N, R281K, N286D, N286Q, and D320N were expressed and assayed. The specific activities of T148G, T148S, R281N, R281K, N286D, N286Q, and D320N are 76.34%, 88.62%, 12.50%, 11.93%, 30.84%, 24.57%, and 48.60% to that of wild-type E3, respectively. The FAD content analysis indicated that these mutant E3s about 71%, 92%, 96%, 93%, 96%, 99%, and 82% of FAD content compared to that of wild-type E3, respectively. The molecular weight analysis showed that these seven mutant proteins form the homodimer. Kinetic's data demonstrated that the Kcat of both forward and reverse reactions of all mutants, except T148 mutants, were decreased dramatically. The results of kinetic study suggest that T148 is not important to E3 catalytic function and the others play a role in the catalytic function of the E3.