劉德中Liu, Te-Chung陳姵如Chen, Pei-RuPei-RuChen營養科學研究所2026-03-242005-01-012000https://handle.ncl.edu.tw/11296/nsktgdhttps://scholars.csmu.edu.tw/handle/123456789/6628V-188及V-243在人類雙羥擬脂醯胺去氫酶反應機制中扮演的角色 中文摘要 酮酸去氫酶複合體是由酮酸去氫酶(α-keto acid dehydrogenase; E1)、雙羥擬脂醯胺醯基轉移酶(dihydrolipoamide acyltransferase; E2)及雙羥擬脂醯胺去氫酶(dihydrolipoamide dehydrogenase; E3)等三種酵素依特定比例所共同組成的複合體。哺乳類的酮酸去氫酶複合體包括：(1)丙酮酸去氫酶複合體( pyruvate dehydrogenase complex, PDC)；(2)α-酮戊二酸去氫酶複合體(α-ketoglutarate dehydrogenase complex, KGDC)；(3)側鏈酮酸去氫酶複合體( branched chainα-keto acid dehydrogenase complex, BCKADC )。其中酮酸去氫酶(α-keto acid dehydrogenase; E1)與雙羥擬脂醯胺醯基轉移酶(dihydrolipoamide acyltransferase; E2)在以上三個酮酸去氫酶複合體中分別不同，而雙羥擬脂醯胺去氫酶(dihydro- lipoamide dehydrogenase；E3)，則是上述三者的共同組成部份。 酮酸去氫酶複合體位於粒線體(mitochondria)內，由於在人體營養之能量中間代謝上催化以下反應： α-keto acid ＋ CoA ＋ NAD+  CO2 ＋ acyl CoA ＋ NADH ＋ H+ 故當共同組成部份(即E3)功能缺乏時會因此引起一些缺陷症，可見E3在醣類、胺基酸及脂肪酸之能量中間代謝上扮演著非常重要的角色。 本實驗即以E3突變蛋白進行研究，藉由V-188及V-243兩個位置之突變蛋白質研究結果，可以對E3反應機制有進一步的了解，以及釐清此兩個胺基酸在電子傳遞路徑上所扮演的角色。在方法學上是使用定點突變方法(SDM)，分別在E3蛋白質上第188個胺基酸(Valine)或第243個胺基酸(Valine)製造突變，以產生突變蛋白質，並利用FAD含量測定、分子量測定、酵素動力學分析、氧化還原中點電位測定、可見光/紫外光光譜分析及螢光光譜分析等方法，來探討酵素結構與功能間的關係。 以定點突變方法構建了四個較穩定的突變蛋白質，分別為V188A、V188G、V188S(E3蛋白質上第188個胺基酸由V變成A、G、S)及V243A(E3蛋白質上第243個胺基酸由V變成A)。由純化分析表得知，突變蛋白質的比活性皆降低。由FAD含量測定結果得知，突變蛋白質FAD含量比卻不受重大影響，代表這兩個胺基酸不參與FAD與蛋白質的結合，亦代表FAD含量不是突變蛋白質比活性降低的主要原因。經由分子量測定發現突變蛋白質與正常蛋白質其分子量大致相同，約為102 kDa；由此顯示突變並不會影響其同質雙體(homodimer)的結構。雖然由酵素動力學分析(enzyme kinetic analysis)的結果顯示突變蛋白質及E3的反應機制仍屬於雙受質乒乓機制( 2 substrates ping-pong mechanism)。然而突變蛋白質酵素反應之代謝迴轉速率(Kcat)都比E3為低。由氧化還原中點電位測定結果發現，active disulfide的中點電位並未有重大改變，此代表active disulfide之還原不受突變影響。但是突變蛋白質的FAD中點電位都增加，此代表電子不易傳遞，也就是FAD還原受突變影響。而由可見光/紫外光光譜分析及螢光光譜分析測定應證上述結果，突變後酵素功能雖然正常，仍可由E form還原成EH2 form，再由EH2 form氧化成E form，此變換週期期間也會產生charge transfer的過渡狀態，但由突變蛋白質之變換週期時間延長可推測，FAD還原形成FADH+ H+，再進一步的將NAD+還原產生NADH，而FADH +H+會再氧化形成FAD的速率變慢，代表突變蛋白質的效率較正常蛋白質的效率為低。 綜合以上實驗結果，V-188、V-243突變後，電子由FAD傳至NAD+的路徑受影響。證實E3上的這兩個胺基酸(V-188、V-243)乃扮演穩定FAD還原電位以及FAD與NAD+間interaction的角色，並參與E3之FAD與NAD+間的電子傳遞。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.雙羥擬脂醯胺去氫酶V-188V-243Dihydrolipoamide Dehydrogenase (E3)thesis