| Summary: | 碩士 === 中山醫學院 === 營養科學研究所 === 89 === The purpose of this research is to characterize the functional significance of N-286 (286th Asparagine of E3) in the E3 reaction mechanism. The first step of this study is using site-directed mutagenesis (SDM) to create N-286 mutant proteins and the mutant proteins are subjected into the following analysis : molecular sieving analysis, relative FAD content assay, enzyme kinetic assay, midpoint reduction potential analysis, spectrophotometrical analysis, and fluorescence analysis.
The purification table shows that the specific activity of these mutant enzymes are lower than that of E3.The relative FAD content assay shows the FAD content of mutant proteins only reduced slightly. According to the molecular sieving analysis, we find that the mutant proteins, N286D and N286Q have normal self-dimerization. Their molecular weight are close to 102 kDa which represented an E3 homodimer. The kinetic analysis shows that E3 and the mutant proteins, N286D and N286Q are following two substrates ping-pong mechanism. But the kcat of these enzymes are striking lower than that of E3.
The results of spectrophotometrical analysis and fluorescence analysis reveal that these mutant proteins can transfer the electron from substrate DHL through active disulfide.But the electron transfer from FADH2 to NAD+ was blocked,which cause the reduction of turnover rate.
In conclusion, the N-286 mutant proteins decrease the ability of electrons transferring between FAD to NAD＋. Therefor , N-286 may be involved in the active center of E3 and play an important role in electrons transferring between FAD to NAD+.
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