支鏈型胺基酸合成路徑 (branched chain amino acids biosynthetic pathway) 涉及了三種不同的酵素來參與整個化學反應。酮酸還原異構酶 (ketol-acid reductoisomerase) 是支鏈型胺基酸合成路徑中參與反應的第二個酵素。這個酵素對於開發新型的除草劑與抗生素是一個很好的目標，因為它不存在於人體與動物之中。酮酸還原異構酶的催化總反應依序為，第一步，與鎂離子有關的烷基轉移；第二步，利用菸鹼醯胺腺嘌呤二核苷酸磷酸或菸鹼醯胺腺嘌呤二核苷酸NAD(P)H 進行酮基還原。然而，對於酮酸還原異構酶的反應機制依然是不夠清楚的，而且對於鎂離子在酵素中扮演的角色與總反應的起始劑為何都是不清楚的。在本篇研究中，我們的研究對象為Sso-ilvC2 蛋白，它是硫化葉菌 (Sulfolobus solfataricus) 的酮酸還原異構酶，我們利用分子動態柔性擬合 (molecular dynamic flexible fitting) 技術與低溫電子顯微鏡 (cryo-EM) 影像 (解析度 = 3.4 Å) 來建立Sso-ilvC2 蛋白的三維結構。我們利用密度泛函理論的計算方法來探討，在不同情況下的反應物，對於甲基轉移所需的活化能。在我們的研究中發現，反應物上的酮基先被質子化後，在進行甲基轉移，所需要的活化能是最低的 (21.5 kcal/mol)。

The ketol-acid reductoisomerase (KARI) is an enzyme necessary for the branched chain amino acids (BCAA) biosynthetic pathway. KARIs catalyze an alkyl-migration of substrate followed by a ketol-acid reduction in terms of NAD(P)H. However, the KARI reaction mechanism is remained unclear; in particular, the roles of Mg2+ ion pair in the catalysis and how the KARI reaction is initialized are still unknown. In this study, we obtain the 3D structure of Sso-ilvC2 protein, a KARI from Sulfolobus solfataricus, from the cryo-EM (resolution = 3.4 Å) in terms of the molecular dynamic flexible fitting (MDFF) technique. We employ the first principle DFT calculations to investigate the activation energy of methyl migration under different catalysis conditions of substrate based on the active site structure of Sso-ilvC2 protein obtained from the cryo-EM structure refined by MD simulations. We observed that the substrate when its ketone group is protonated has lowest active energy (21.5 kcal/mol) for the methyl migration.

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