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V188A、V188S及V188G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27.3%、7.9%及1.6%。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101%、95%及97.5%。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V188A正反應的Kcat下降至36.2%而逆反應則維持正常大約為94%。V188S正反應的Kcat下降至9.1%而逆反應則下降為6.4%。V188G正反應的Kcat下降至2.1%而逆反應則下降為9.6%。其中V188A正反應中NAD/sup +/之Km增加大約1.5 fold,V188G正反應中NAD/sup +/之Km增加大約2.8 fold。FAD之氧化還原電位中點則由E3之-314mV上升至-304(V188A)、-300(V188S)及-270(V188G)。此結果說明V188參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, V188A, V188S and V188G, were over-expressed, purified and characterized. The specific activities of both mutant proteins are 27.3%, 7.9% and to that of the wild-type E3. The FAD content analysis indicated that these three mutant E3s have about 101%, 95% and 97.5% 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 mutant proteins were decreased to about 36.2% (V188A), 9.1% (V188S) and 2.1% (V188S). The Kcat of reverse reaction of V188A protein was remain normal (about 94%) while V188S protein was decreased (about 6.4%) and V188G was decreased (about 9.6%) compared to that of wild-type. For V188A and V188G mutant E3s, the Km for NAD/sup +/ in the forward reaction increased by about 1.5 and 2.8 fold. The mid-point oxidation-reduction potential of mutant proteins were increased from -314mV (E3) to -304 (V188A), -300 (V188S) and -270 (V188G). It suggests V188 involved in electron transfer between FAD and NAD/sup +/ of the catalytic function of the enzyme.
