為了探討二氫硫辛酸去氫@@(E3)的反應機制,反以本研究利用定點突變的方式創造出兩種E3的突變蛋白質,分別是R281K及R281N經大量表達此三突變蛋白質以及純化後發現其專一性的活性分別是正常E3的5.4%及12.5%。經FAD含量分析後發現其FAD含量並沒有因為突變取代而大量降低,分別是正常E3的100%及96.4%。分子量分析的結果顯示,突變蛋白仍保持其具有生物活性的雙體構造。動力學的研究顯示R281K正反應的Kcat下降至44%而逆反應則下降至39%。R281N正反應的Kcat下降至44%而逆反無下降。FAD之氧化還原電位中點則由E3之-314mV上升至-280(R281K)及-290(R281N)。此結果說明R281參與酵素的正逆反應中電子由FAD轉移至NAD/sup +/之機制。
To investigate the reaction mechanism of human dihydrolipoamide dehydrogenase (E3), three mutant human E3s, R281K and R281N, were over-expressed, purified and characterized. The specific activities of both mutant proteins are 5.4% and 12.5% to that of the wild-type E3. The FAD content analysis indicated that these two mutant E3s about 100% and 96.4% of FAD content compared to that of wild-type E3. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetics data demonstrated that the Kcat of forward reaction of both mutant proteins were decreased to about 44%. The Kcat of reverse reaction of R281K was decreased to about 39%) compared to that of wild-type. The mid-point oxidation-reduction potential (FAD) of mutant proteins were increased from -314 mV (E3) to -280 (R281K) and -290 (R281N). It suggests R281 involved in electron transfer between FAD and NAD/sup +/ of the catalytic function of the enzyme.