| Summary: | 碩士 === 中山醫學院 === 營養科學研究所 === 86 === Mammalian α-keto acid dehydrogenase complexes includes pyruvate dehydrogenase comples (PDC),α-ketoglutarate dehydrogenase (αKGDC),branched chain α-keto acid dehydrogenase complexes(BCKADC). These enzyme complexes catalyze the oxidative decarhboxylation of α-keto acid with the formation of acyl-CoA, CO2 and NADH. The α-keto acid dehydrogenase complexes are found in the mitochondria. Each enzyme complex is composed of three catalytic components: α-keto acid dehydogenase (E1),dihydrolipoamide acyltransferase (E2) and dihydrolipoaimide dehydrogenase (E3). E1 and E2 show substrates specificity and E3 is the common component for all α-keto acid dehydrogenase complexes .In case of E3 deficiency , it will affect all α-keto acid dehydrogenase complexes.
Since E3 plays an important role in the energy metabolism. The approach of this study is characterizing the electron transferring pathway of E3 by using site-directed mutagenesis (SDM).
To investigate the role of lysine-54 of human dihydrolipoamide dehydrogenase (E3) in the formmation of intermediate during electrons transfer two mutant human E3s, S53K54-K53S54 (SK-KS) and K54E, were overexpressed, purified and characterized. Molecular sieving analysis showed that each proteins (E3, SK-KS, K54E )was dimer with the molecular weight approximately to 100 kDa. The results of kinetic analysis show the E3 and ES-KS following two substrates'' ping pong mechanism, The relative FAD content of the enzymes is E3:SK-KS:K54E=100:82:90 .The relative specifc activity of the enzymes is E3:SK-KS:K54E=100:46:1.7.The fluorescence analysis show that E3'' FAD can be reduced by DHL and then formed FADH.SK-KS mutant''s FADH reoxidation is significantly slowly than the wild type E3 and K54E mutant''s FAD is extremely could not be reduced by DHL. The FADH will be oxidized by NAD+with the NADH formation, At this stage, the mutant proteins (SK-KS and K54E) are significantly slowly than the wild type E3 .The data showed that the number 54th amino acid (K54) of E3 cDNA maybe participate in the enzyme''s active center and specifically significantly corresponding for enzyme''s activity.
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