R-281與D-320在人類雙羥擬脂醯胺去氫反應機制中所扮演的角色

摘 要 本實驗的目的是要研究酮酸去氫複合體中，雙羥擬脂醯胺去氫(dihydrolipoamide dehydrogenase; E3)的反應機制；哺乳類的酮酸去氫複合體包括丙酮酸去氫複合體(pyruvate dehydrogenase complex, PDC)、α-酮戊二酸去氫複合體(α-ketoglutarate dehydrogenase complex, KGDC)及側鏈酮酸去氫複合體(branched-chain α-keto acid dehydrogenase complex, BCKADC)等三種複合體。酮酸去氫複合體位於粒腺體，具有酮酸去氫(α-Ketoacid dehydrogenase；E1)，雙羥擬脂醯基轉移(dihydrolipoamide acyltransferase；E2)及E3進行催化反應。其中E1和E2具受質專一性(substrate specificity)。而E3是此三種酵素複合體的共同組成部份。因此E3的酵素活性和此三種酵素複合體的酵素活性有相關性。當E3缺乏時三種酮酸去氫複合體都會受到影響。典型的症狀包括乳酸血酸(lactic acidosis)，智障，血清中氨基酸濃度增加及升高尿液中有機酸濃度。由此可知，E3在營養學上有關醣類、氨基酸、脂肪之能量中間代謝上佔有舉足輕重的地位，而且人類E3缺乏後會產生嚴重的代謝疾病。 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102kDa；換言之突變並不會影響其同質雙體(homodimer)的結構。酵素相對的FAD含量比E3：R281N：R281K：D320N為100：96：100：83。而由酵素動力學分析(enzyme kinetic analysis)的結果顯示R281N、R281K、D320N及E3的反應機制是屬於雙受質乒乓機制(2 substrates ping-pong mechanism)。酵素反應之代謝迴轉速率(Kcat)比E3：R281N：R281K：D320N為100：43：44：19.2。光譜分析及氧化還原中點電位測定，結果發現R281N之Active Disulfide還原電位變高，所以有charge transfer之形成。而R281K之FAD還原電位變高，所以未有charge transfer形成之現象。D320N也未有charge transfer形成，同時無法將FAD還原，且無NADH的產生。 綜合以上實驗結果：R-281主要是穩定FAD的構造，以利於電子傳遞至FAD。D-320則是電子從FADH往下傳遞至NAD+之重要關鍵胺基酸。

Abstracrt by Shing-Ling Chang The mammalian α-ketoacid dehydrogenase complexes include pyruvate dehydrogenase (PDC) , α-ketoglutarate dehydrogenas(KGDC), and branched-chain α-ketoacid dehydrogenas complexes (BCKADC). Each enzyme complex is composed of three catalytic components,namely, α-ketoacid dehydrogenase (E1), dihydrolipoamide acyltransferase (E2), and E3.E1 and E2 show substrate specificity and E3 is the common component for all α-ketoacid dehydrogenase complexes. A deficiency in E3 leads to the deficiency of all these threeα-ketoacid dehydrogenase complexes. The symptoms of E3 deficiency include lactic acidosis, neurological dysfunction, increased serum amino acids and increased urinary organic acids. Since E3 plays an important role in the intermediary metabolism and in case of E3 deficiency, the patient will suffer serious metabolic disorder. The objective of this experiment is to study the reaction mechanism of dihydrolipoamide dehydrogenase(E3). Molecular sieving analysis showed that the R281N，R281K and D320N mutant protein was homodimer with the molecular-weight approximately to 102 kDa, as well as wild-type E3. The relative FAD content of these enzymes are E3：R281N：R281K：D320N＝100：96：100：83 . Kinetic analysis show the E3 and three mutant proteins, R281N、R281K and D320N, following two substrates ping-pong mechanism. The Kcat of these enzymes are E3：R281N：R281K：D320N=100：43：44：19.2 . Spectrophotometer analysis and midpoint reduction potential analysis show that mutant protein (R281N) of active disulfide midpoint reduction potential increased. The mutant protein (R281K) of FAD midpoint reduction potential increase and have not charge transfer. The mutant protein (D320N) reveals have not charge transfer and the FAD can not be reduced . In conclusion, the R281N and R281K may influence the charge transfer formation. The D320N may influence the ability of electrons transferring between FAD to NAD+.