The role of E-192 in the Reaction Mechanism of Human Dihydrolipoamide Dehydrogenase(E3)

• Main Authors: Chin-Yin Shih, 施智尹
• Other Authors: Te-Chung Liu
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/88099377807089518314

碩士 === 中山醫學院 === 營養科學研究所 === 87 === Dihydrolipoamide dehydrogenase(E3) belongs to the enzyme family of pyridine nucleotide-disulfide oxidoreductases and catalyzes the transfer of electrons from dihydrolipoamide to FAD cofactor , then to the NAD＋. E3is also the common component of α-ketoacid dehydrogenase complexes including pyruvate dehydrogenase (PDC) , branched-chain α-ketoacid dehydrogenas complexes and α-ketoglutarate dehydrogenas complexes. A deficiency in E3 leads to the deficiency of all these threeα-ketoacid dehydrogenase complexes. E3 plays an important role in the energy metabolism indeed. This study is to characterize the functional significant of E-192 in the reaction mechanism and to focused on the role of E-192 in electron transferring pathway. The approach in this study was using site-directed mutagenesis(SDM) to create the E192Q mutant protein, and the mutant protein was subjected into the following analysis which including molecular sieving analysis, relative FAD content assay , enzyme kinetics assay , spectrophotometer analysis and midpoint reduction potential analysis. Molecular sieving analysis showed that the E192Q mutant protein was homodimer with the molecular-weight approximately to 102 kDa, as well as wild-type E3. The relative FAD content of these enzymes are E3：E192Q＝100：95.The Kcat of E192Q is reduced to 5%, while compared to wild-type E3. It is interesting that the reaction mechanism of E192Q is sequential mechanism while E3 is the ping pong mechanism in forward reaction. The fluorescence assay reveals that the FAD can be reduced by DHL with the formation of FADH in E3. The FADH can be further oxidized by NAD＋with the NADH formation. E192Q almost loss the ability of electrons transferring between FAD to NAD＋when compared to E3. In addition, the UV/VIS spectrophotometer analysis of E192Q indicates that the electron transferring from active disulfide to FAD may be blocked. Further, the midpoint potential of FAD is also increased. In conclusion, the E192 maybe involve in the active center of E3 and specifically corresponding to the reduction of FAD in the E3.reaction mechanism.