Abstract
解答白化綠豆下胚軸H-ATPase(E.C.3.3.1.3)與H-PPase( E.C.3 .3.1.1)兩種能量酵素結構與功能.我們利用(1)化學修飾法(2)螢光分析 法(3)熱力學 ( 4)瑩光能量轉 )輻射鈍化法來探知.標的物, 例如, sulhydryl, tyrosyl, histidyl和lysinyl專一藥物和受質的相似物,用以 共價結合在ATPase或者PPase活化中心上的必需氨基酸.所有的標的物均能 引發酵素活性 (含受質水解與質子傳遞)喪失. 均坐落在 A次單元體上,且 生理受質或其他受質相似物均有不同程度的保護作用.由時間與酵素活性 之間作雙對數與雙倒數圖形,與結合比率實驗指出至少有一個必須氨基酸 存在活化中心內.並由此結果推出核甘酸與必須氨基酸的相關位置.由內在 蛋白質螢光用acrylamide,iodide,nitrate及chl oride觀察芬香族群的環 境與結構之改變.我們推論大部分的芳香氨基酸是包埋在整個酵素內部,且 生理抑制離子nitrate與生理刺激離子chloride作用機制是不同的而引起 不同的生理影響.再由熱力學實驗得知,在不同溫度下酵素存在至少二種形 態.活化能在 20KJ/mol且受ATP/Mg保護的ATPase則會阻止熱抑制發生,活 化能在7KJ/mol但先受FITC作用的ATPase則會促進熱抑制的發生,活化能 在26KJ/mol可見不同物質處理所產生不同程度的結構改變對熱抑制有不同 的感受度.利用IAEDANS與FITC分別用作為提供者與接受者來決定活化中心 上必須cysteine及lysine之空間距離,得到光譜重疊面積是1.5x10cm. MFoster距離是29o,能量傳遞效率是 58%,而求得空間距離是27o.我們也撿 視了不同標的物對PPase生理活性的影響.值得注意的是抑制百分五十濃度 在液泡中膜上質子傳遞與純化出PPase水解受質能力有極大的不同.暗示著 這二種生理功能並非直接相連著且個別單元體具獨立水解受質能力,並受 膜電位差來調控著. All these probes caused marked inactivation of enzyme activities of both membrane-bound and solubilized ATPases and its associated protontranslocation.Thebin ding sites were shown to locateat catalytic domain of A TPase by protection and labeling studies.Thedouble-loga rithmic plots of apparent rate constantsversus modifier concentrations gave slopes of approximately one indica ting that atleast one copy ofessential lysine, cystein e, and arginine residue were located at the active site of the catalytic subunit of tonoplast ATPase. A tentat ive architecture at nucleotide binding site of vacuolar ATPase was proposed based on these results. From the quenching parameters,we demonstrated that majo rities of aromatic groups were buried in a relatively h ydrophobic core. From thermodynamic studies,there are a t least two conformational states for ATPase. Substrate -binding of ATPase stabilized the enzyme structureby in crease intheactivation energy from 20.6to 25.9KJ/mol. W e believed that the conformation of the enzyme was alte red to protect against the thermoinactivation. In contr ast,the modification of ATPase byfluorescein 5'-isothio cyanate could accelerate theinhibition by heat. The spa tial arrangement and the relationship of structure and function in the catalytic region ofATPase were studied.