- 作者: Yi-Chun Wang; Shih-Tsung Wang; Chuan Li; Wen-Hu Liu; Pei-Ru Chen;Ling-Yun Chen; Te-Chung Liu
- 作者服務機構: 1 institute of Medicine, Chung Shan Medical University, Taichung, 402, Taiwan, ROC; ; 2 Department of Biomedical Sciences, Chung Shan Medical University, Taichung , 402, Taiwan, ROC; ; 3 Department of Nutritional Science, Chung Shan Medical University, No.110, Sec. 1, Chien-Kuo N. Rd, Taichung , 402, Taiwan, ROC; ; 4 Department of Radiation Safety Room, Chung Shan Medical University, Taichung, 402, Taiwan, ROC; ; 5 Department of Instrument Center, Chung Shan Medical University, Taichung, 402, Taiwan, ROC; ; 6 Department of Biochemistry, Chung Shan Medical University; Taichung , 402, Taiwan, ROC
- 中文摘要: --
- 英文摘要: According to the multiple alignment of various dihydrolipoamide dehydrogenases (E3s) sequences, three human mutant E3s of the conserved residues in the center domain, N286D, N286Q, and D320N were created, over-expressed and purified. We characterized these mutants to investigate the reaction mechanism of human dihydrolipoamide dehydrogenases. The specific activities of N286D, N286Q, and D320N are 30.84%, 24.57% and 48.60% to that of the wild-type E3 respectively. The FAD content analysis indicated that these mutant E3s about 96.0%, 99.4% and 82.7% of FAD content compared to that of wild-type E3 respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic's data demonstrated that the Kcat of both forward and reverse reactions of these mutant proteins were decreased. These results suggest that N286 and D320 play a role in the catalytic function of the E3.
- 中文關鍵字: --
- 英文關鍵字: Dihydrolipoamide Dehydrogenase, enzyme kinetics, site-directed mutagenesis