酚亞硫酸基轉移酵素之機制探討 : 氧化還原與核苷酸之影響

Abstract

酚亞硫酸基轉移酵素需要adenosine 3',5'-bisphosphate (PAP) or 3'-phospho adenosine 5'-phosphosulfate (PAPS) 當成輔因子或輔受質，進行亞硫酸基催化反應。本論文內容包含四部份：首先建立新的分析方法，測量PAP或PAPS於picomole範圍及分析PAPS純度；進一步決定豬肝、鼠肝、大腸桿菌中PAP量。第二、我們得知ribose和adenine是輔因子與酵素結合上主要兩區域。從解離常數發現許多不同種類核苷酸也能緊密地與酵素結合；其中adenosine 5'-monophosphate, adenosine 2',5'-bisphosphate and adenosine 2':3'-cyclic phosphate 5'-phosphate可以取代PAP進行催化反應。並利用光譜儀、HPLC、31P-NMR觀察亞硫酸基轉移活性與最終產物；測量酵素Vmax和輔因子或輔受質進行亞硫酸基轉移之Km。第三、探討酚亞硫酸基轉移酵素中雙硫鍵調節酵素功能之角色。用氧化還原試劑處理，發現只有同時存在66與232位置時，PAP與酵素間解離常數會有明顯程度變化。測量酵素Vmax和PAP之Km，於不同氧化還原狀態下之影響；但突變66與232位置，仍發現微小程度改變。證實66與232位置為調節酚亞硫酸基轉移酵素氧化還原之活性，是經由控制核苷酸結合或釋放所導致。最後研究核苷酸在不同氧化還原狀態效果，發現PAP和N6-(6-aminohexyl) adenosine 3',5'-diphosphate活化及穩定酚亞硫酸基轉移酵素。利用5,5'-Dithiobis (2,2'-nitrobenzoate)測量酚亞硫酸基轉移酵素中半胱胺酸氧化程度。於初步氧化時活性不受影響，此時有兩個半胱胺酸被氧化；進一步氧化使酵素失去活性。

Sulfuryl group transfer catalyzed by phenol sulfotransferase (PST) requires adenosine 3',5'-bisphosphate (PAP) or 3'-phospho adenosine 5'-phosphosulfate (PAPS) as cofactor or cosubstrate, respectively. Firstly, this work presents novel colorimetric methods not only to measure PAP and PAPS in the range of picomoles, but also to determine the purity of PAPS or PAP contaminants in PAPS in the range of nanomoles. Furthermore, this method is used to determine the amount of PAP in extracts of pig liver, rat liver, and Escherichia coli. Secondly, we find that ribose and adenine, two major parts of the adenosine nucleotide, bind tightly to PST separately, and various nucleotides also bind tightly to PST. We determine the dissociation constants of a variety of nucleotides and examine their potential as cofactors or cosubstrates of PST. Adenosine 5'-monophosphate, adenosine 2',5'-bisphosphate and adenosine 2':3'-cyclic phosphate 5'-phosphate, are shown to be sulfated at 5’-phopho position by a PST catalyzed reaction. Spectrophotometry, HPLC, and 31PNMR are used to determine the activity of PST and identify the sulfated nucleotides. The Vmax of PST and Km of these nucleotides are determined when they are used as cofactors or cosubstrates for the sulfuryl group transfer. Thirdly, we study the role of disulfide bonds in regulating enzyme function. In different redox condition, by treating the protein with GSSG or Tris (2-carboxyethyl) phosphine, the dissociation constants of PAP and PST may differ four orders of magnitude but only in the presence of both C66 and C232. Km (PAP) and Vmax of the PST and its cysteine mutants are affected in different redox condition, but are less significant for those mutants whose C66 and C232 were mutated. We conclude that Cys66 and Cys232 regulate the activity of phenol sulfotransferase with redox environment through the control of nucleotide binding and release. Fnally, we investigated the effect of nucleotides, PAP and N6-(6-aminohexyl) adenosine 3',5'-diphosphate, on the activity and stability of PST in different redox status. Using 5,5'-Dithiobis (2,2'-nitrobenzoate) to react with the PST and its mutants, we are able to identify the order of oxidation on each cysteine residue. Initial oxidation of PST does not affect the activity of PST. In this stage only two cysteines are oxidized. Further oxidation of PST results in the loss of enzyme activity.