研究二氫硫辛酸去氫酵素(E3)P453-L,E192K和W197-F突變蛋白質構造之穩

哺乳動物的酮酸去氫複合體包括丙酮酸去氫 (pyruvate dehydrogenase ，PDC)，酮戊醯酸去氫 (a-ketoglutarate dehydrogenase ，KGDC) 及側鏈酮酸去氫複合體 (branched-chain a- ketoacid dehydrogenase complexes，BCKADC) 三種。酮酸去氫複合體 位於粒線體，具有酮酸去氫 (E1， a-ketoacid dehydrogenase)，二氫 硫辛酸醯基轉移 (E2，dihydrolipoamide acyltransferase) 及二氫硫 辛酸去氫 (E3， dihydrolipoamide dehydrogenase) 以進行催化反應 。由於 E3 是酮酸去氫複合體的共同組成部分，因此當 E3 缺乏時，三 種酮酸去氫複合體均會到影響。E3 缺乏的典型症狀包括乳酸血症 (lactic acidosis )，神經功能失調，血清氨基酸濃度增加等現象。E3 缺陷症是一種遺傳疾病，在 E3 缺陷病人的 E3 cDNA 上曾發現兩個點突 變，其中一個突變是由 C 變成 T，造成蛋白質上第453 個氨基酸由 P 變 成 L (E3 P453→L) 。由於 E3 在能量代謝上扮演很重要的角色，缺乏 E3 會造成嚴重的代謝疾病，因此本實驗的目的即是探討 E3 的突變蛋白 質 P453→L 突變後的變化。此外，亦對氨基酸 E192 及 W197F 在突變後 結構上的穩定性作初步的觀察。 將 E3 的突變蛋白：E192→K ，W197→F 及 P453→L，經隔夜 induction 作用後，經 SDS-PAGE 及 western blot analysis 結果顯示 E192→K 及 W197→F 在突變後表現出degradation form。而P453→L 經 HPLC 分析亦 出現monomer，顯示突變後的蛋白變得較不穩定。根據實驗結果得知：氨 基酸E192，W197，P453 突變後均會造成蛋白質不穩定的現象。

The mammalian a-ketoacid dehydrogenase complexes include pyruvate dehydrogenase (PDC), a-ketogiuatarate dehydrogenase (KGDC) and branched a-ketoacid dehydrogenase complexes (BCKADC). Each enzyme complex is composed of three catalytic components: a-ketoacid dehydrogenase (E1), dihydrolipoamide acyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). E1 and E2 show substrate specificity and E3 is the common component for all a-ketoacid dehydrogenase complexes . A deficiency in E3 lead to the deficiency of all these a-ketoacid dehydrogenase complexes. The feature of E3 deficiency include lactice acidosis, neurological dysfunction, increased serum amino acids and increased urinary organic acid. Two natural mutation in the E3 deficient patient had be identified. One identified mutation changed a single nucleotide from C to T, resulting in the substituon of L (CTG) for P453 (CCG)(E3 P453 to L). Since E3 plays important roles in the energy metabolism and in case of E3 deficiency, the patient will suffer serious metabolic disorder. Therefore, the goal of this experiment is that studies the changes of E3 mutant protein P453→L and the structural stability of mutant protein W197→F, E192→K. By using of SDS-PAGE and western blot analyses, W197-F and E192- K mutant protein showed degradation form. By using of molecular sieving analysis, P453-L showed 75 % is dimer and 25 % is monomer. These results demonstrated that mutant proteins P453-L, W197-F and E192-K were more unstable.