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N286D及N286Q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29.3％及23.8％。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95.1％及99.3％。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N286D正反應的Kcat下降至31.4％。N286Q正反應的Kcat下降至23％。此結果說明N286在酵素的催化反應中扮演一個重要的角色。
To investigate the reaction mechanism of human dihydrolipoamide dehydrogenase (E3), three mutant human E3s, N286D and N286Q, were over-expressed, purified and characterized. The specific activities of both mutant proteins are 29.3% and 23.8% to that of the wild-type E3. The FAD content analysis indicated that these two mutant E3s about 95.1% and 99.3% 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 31.4% and 23%, respectively. It suggests N286 playa a role in the catalytic function of the E3.
