Study of Structure and function Relationship of Human Dihydrolipoamide Dehydrogenase

• Main Authors: Yi-Chun, 王怡鈞
• Other Authors: Ling-Yun Chen
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/19493494684740937247

博士 === 中山醫學大學 === 醫學研究所 === 95 === Human dihydrolipoamide dehydrogenase (hE3) is a common component of α-ketoacid dehydrogenase complexes. E3 belongs to the enzyme of pyridine nucleotide-disulfide oxidoreductases. These enzymes have an active disulfide, an active base, and FAD to transfer electrons between the electron acceptor (NAD+ or NADP+) and dithiol substrate in the active site. Mutations to this homodimeric protein cause E3 deficiency and always fatal. To investigate its reaction mechanism, we first performed multiple sequence alignment with other seventeen eukaryotic E3s. According to hE3 structure and the result of multiple sequence alignment, four amino acids T148, R281, N286, and D320 were selected and subjected into mutagenesis. Seven recombinant mutant E3, T148G, T148S, R281N, R281K, N286D, N286Q, and D320N were expressed and assayed. The specific activities of T148G, T148S, R281N, R281K, N286D, N286Q, and D320N are 76.34%, 88.62%, 12.50%, 11.93%, 30.84%, 24.57%, and 48.60% to that of wild-type E3, respectively. The FAD content analysis indicated that these mutant E3s about 71%, 92%, 96%, 93%, 96%, 99%, and 82% of FAD content compared to that of wild-type E3, respectively. The molecular weight analysis showed that these seven mutant proteins form the homodimer. Kinetic''s data demonstrated that the Kcat of both forward and reverse reactions of all mutants, except T148 mutants, were decreased dramatically. The results of kinetic study suggest that T148 is not important to E3 catalytic function and the others play a role in the catalytic function of the E3.