酚亞硫酸基轉移酵素與Adenosine 3',5'-bisphosphate及其類似物反應之探討

Abstract

本論文始於 adenosine 3*,5*-bisphosphate (PAP) 與酚亞硫酸基轉移酵 素 (phenol sulfotransferase) 關係的研究，內容包含四部份：純化大 量的純化酵素、建立酵素輔因子 (cofactor) 之分析的方法、探討酵素催 化反應中PAP所扮演的角色，並利用紫外光／可見光光譜儀、螢光光譜儀 求得PAP及其類似物之Km、Vmax及KD值，同時以31P-NMR觀察亞硫酸基轉移 到PAP及其類似物 (analogues) 上之現象。針對PAP之分析，我們可以準 確地測出picomole (10-12 mole) 範圍的PAP量。由於此方法的靈敏度相 當高，並且不易受其他雜質的干擾，所以日後與亞硫酸基轉移酵素有關的 分析，均可以利用此方法來測定。我們同時應用此方法，發現在幾種不同 的生物樣品材料中，PAP的含量均遠高於亞硫酸基轉移酵素的含量。由KD 值，我們發現酚亞硫酸基轉移酵素可以與許多種PAP及其類似物結合，且 有多重選擇性；而在活性催化反應上則有相當嚴格的選擇性。我們更進一 步分析PAP及其類似物官能基之結構與酵素結合及活性之關係，發現 5*-Phosphate是活性反應上必要的結構，而Ribose和Adenine是輔因子與 酵素結合上主要的兩個部分。 This research concerns the effect of adenosine 3',5'-bisphosphate (PAP) on the function of phenol sulfotransferase. Approaches for this research include four steps. Firstly, prepare a large amount of the purified enzyme. Secondly, establish analytical methods of PAP and 3*-phospho- adenosine- 5*-phosphosulfate (PAPS). Thirdly, study the role of PAP in the phenol sulfotransferase catalyzed reaction . Lastly, determine Km, Vmax, KD of PAP and its analogues by UV/VIS and fluorescence spectrophotometry and We have developed a method for PAP determination in the range of picomole (10-12 mole). Since the sensitivity of the method is very high, and it is not easy to get interfered by other impurities, it can be a standard procedure for the measurement of PAP. We have determined that the content of PAP is much higher than that of phenol sulfotransferase in several biological species.Binding of PAP and its analogues by phenol sulfotransferase is rather non- selective. We have determined binding constants of variety of PAP analogues and examined their potential as cofactors or substrate of phenol sulfotransferase. Structure and functional groups of PAP analogues and their requirements for phenol sulfotransferase binding have been examined. We have found that even ribose and adenine, two major parts of the adenosine nucleotides, tightly bound to phenol sulfotransferase separately.