| Summary: | 碩士 === 中山醫學院 === 營養科學研究所 === 88 === Characterization of V-188 and V-243 in the Reaction Mechanism of Human Dihydrolipoamide
Dehydrogenase (E3)
Abstract
by
Chen, Pei-Ru
α-keto acid dehydrogenase complexes is composed of three catalytic components：α-keto acid dehydrogenase (E1), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). Mammalian α-keto acid dehydrogenase complexes includes：(1) pyruvate dehydrogenase complexes (PDC)；(2)α-keto-glutarate dehydrogenas complexes (KGDC)；(3)branched chainα-keto acid dehy-drogenas complexes (BCKADC). Dihydrolipoamide dehydrogenase (E3) is the common component for all α-keto acid dehydrogenase complexes.
These enzyme complexes are in mitochondria, and catalyze the oxidative decarboxylation of α-keto acid with the formation of acyl-CoA, CO2 and NADH.
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.
The research goal is to characterize the functional significance of V-188 and V-243 (188th Valine of E3 ,243th Valine of E3) in the E3 reaction mechanism.The approach in this study was using site-directed mutagenesis (SDM) to create V-188 and V-243 mutant proteins and the mutant proteins were subjected into the following analysis which including relative FAD content assay, molecular sieving analysis, enzyme kinetic assay, midpoint reduction potential analysis, spectrophotometrical analysis, and fluorescence analysis.
All the specific activity of these mutant enzymes are lower than that of E3, and the relative FAD content does not be effected seriously. V188A, V188G, V188S, and V243A have normal self-dimerization, and their molecular weight were about 102 kDa which represented an E3 homodimer. Kinetic analysis show the E3 and the four mutant proteins, V188A, V188G, V188S, and V243A, following two substrates ping-pong mechanism. The Kcat of these enzymes are lower than E3.
Midpoint reduction potential analysis show that all mutant proteins have no serious change on the midpoint reduction potential of active disulfide.But all mutant proteins have been effected on the midpoint reduction potential of FAD. The midpoint potential of FAD of all mutant proteins are increased.
The results of spectrophotometrical analysis and fluorescence analysis show that all mutant proteins have normal function, however, the efficiency of electron transfer from FAD to FADH+H+, from FADH+H+ to NAD+, and from NAD+ to NADH is decreased, comparing with E3, in the mutant proteins.
In conclusion, the ability of electrons transferring between FAD to NAD＋of V-188 and V-243 may be effected when compared to E3. Therefore, V-188 and V-243 may be involved in the active center of E3 and play an important role of the electron transferring between FAD to NAD＋in the E3. reaction mechanism.
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