醇亞硫酸基轉移酶及3'磷酸腺甘酸5'磷酸硫酸合成酶之螢光檢測法

Abstract

亞硫酸化普遍地存在於生物系統中，而且被亞硫酸化的生物分子會明顯改變其生理化學性質。過去的研究已經證實3′磷酸腺甘酸5'磷酸硫酸合成酶(3’-phosphoadenosine 5’-phosphosulfate synthetase, PAPSS)和亞硫酸基轉移酵素(sulfotransferase, ST)兩類酵素涉及亞硫酸化反應。前者可活化無機硫酸根成為PAPS，後者則催化產生的PAPS之亞硫酸基至接收分子。本論文主要是發展螢光檢測法來探討此兩類酵素在亞硫酸化的重要性，如探討四級結構對於亞硫酸基轉移酵素的影響，並分成三個章節敘述之。首先，我們建立了不同類的亞硫酸基轉移酵素的表達與純化方法。而且首次報導關於醇亞硫酸基轉移酵素(alcohol sulfotransferase, AST)之螢光檢測法。此方法有別於放射性同位素的終點式檢測，屬於連續性的螢光檢測法。檢測系統的主旨是利用重組的酚亞硫酸基轉移酵素(phenol sulfotransferase, PST)催化受質4-methylumbelliferyl sulfate (MUS)做為亞硫酸基提供者並轉移至3’-phosphoadenosine 5’-phosphate (PAP)再生3’-phosphoadenosine 5’-phosphosulfate (PAPS) ，此核苷酸的亞硫酸基再由AST催化。在設計的環境中，重組的PST不會催化AST的受質，而且反應後產生4-methylumbelliferone (MU)之螢光變化不但可以定量AST的活性，也有足夠的靈敏度來測出ng或pmole的酵素活性。在此研究中，許多hDHEA-ST的生化特性可被測得，文中並將此法與放射性同位素法就靈敏度等特質相比較，以及於第二章中研究四級結構對於亞硫酸基轉移酵素的影響。第三章，我們成功地利用分生技術得到重組的3′磷酸腺甘酸5'磷酸硫酸合成酶。並偶合使用亞硫酸基轉移酵素的生理反應條件，開發螢光檢測法以測量出3′磷酸腺甘酸5'磷酸硫酸合成酶的活性。此兩套AST及PAPSS之螢光檢測法不僅對於常規且詳細的酵素動力學有幫助外，也有潛力利用微量盤成為高效能分析法。

The sulfoconjugation of biomolecules occurred widely in biological system which resulted in a dramatic change in the physicochemical property of the sulfonated compounds. Previously, 3’-phosphoadensine 5’-phosphosulfate synthetase (PAPSS) and sulfotransferases (STs) were found to involve in the course of sulfonation. The former activates inorganic sulfate to form PAPS, and the latter catalyzes the sulfuryl group of PAPS transferring to an acceptor molecule. The thesis is divided into three chapters, and introduces sensitive fluorometric assays developed for investigation into their fundamental issues concerning the role of sulfonation, such as the effects of quaternary structure on sulfotransferase function. First chapter described the convenient expression and purification procedure of recombinant STs by affinity column. Furthermore, a first continuous fluorometric assay was developed for alcohol sulfotransferase (AST). It utilized PAPS regenerated from 3’-phosphoadenosine 5’-phosphate (PAP) by a recombinant phenol sulfotransferase (PST) using 4-methylumbelliferyl sulfate (MUS) as the sulfuryl group donor. The change of fluorescence intensity of 4-methylumbelliferone (MU) corresponded directly to the amount of active AST and was sensitive enough to measure ng or picomole amount of the enzyme activity. Some properties of hDHEA-ST were determined by this method and were found comparable to published data. In second chapter, this coupled-enzyme assay was applied to study some effects of quaternary structure on AST. Some catalytic properties and thermal stability were compared between dimeric (wild-type) and monmeric (mutant) cytosolic STs. Final chapter described molecular cloning, expression, and purification of recombinant PAPSS. By coupling with the physiological reaction of PST, the activities of recombinant PAPSS were also determined fluorometrically. The two fluorometric methods developed not only are useful for the routine and detailed kinetic study of these important class of enzymes but also have the potential for the development of a high-throughput procedure using microplate reader.