N-286在人類雙羥擬脂醯胺去氫酶反應機制中所扮演的角色

本實驗即以E3突變蛋白進行研究，探討N286這個胺基酸在E3反應機制與電子傳遞路徑上扮演的角色。實驗方法首先利用定點突變方法（SDM）構建N286突變蛋白質，即將N（Asparagine）分別突變成D（Aspartic acid）與Q（Glutamine）。接著利用分子量測定、FAD含量測定、酵素動力學分析、螢光光譜分析、可見光/紫外光光譜分析及氧化還原中點電位測定等方法，深入探討此突變蛋白質之酵素結構與功能間的關係。 由初步的酵素活性分析得知，此二種突變蛋白質N286D與N286Q的比活性均降低，分別為E3的29.3%與23.8%。由FAD含量測定結果得知，此二種突變蛋白質N286D與N286Q其FAD含量比並不受突變影響，分別為E3的95.1%與99.3%。由分子量測定發現突變蛋白質與正常蛋白質之分子量大致相同，約為102 kDa；由此可知，N286並不參與同質構造的形成，故將蛋白質突變並不會影響其同質雙體（homodimer）的結構。在酵素動力學分析（enzyme kinetic analysis）的結果顯示突變蛋白質的反應機制與E3相同，均屬於雙受質乒乓機制（2 substrates ping-pong mechanism），但突變後蛋白質其酵素反應之代謝迴轉速率（kcat）均較E3為低。N286D與N286Q之正反應kcat分別為E3的31.4%與23%；其逆反應之kcat分別為E3的25.7%與28.6%。接著利用螢光光譜分析與可見光/紫外光光譜分析，進一步探討酵素突變後對其電子傳遞路徑的影響。由結果得知，突變後蛋白質仍有charge transfer之產生，表示電子可經由DHL傳至active disulfide，但是電子由FADH2傳遞至NAD+的路徑卻發生阻礙，導致酵素代謝迴轉速率下降。 綜合以上實驗結果得知：將N-286突變後，阻礙了電子由FAD傳至NAD+的路徑。由此可知，N286是穩定FAD構造的重要胺基酸之一，並且參與FAD與NAD+之間的電子傳遞。

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+.