探討半胱胺酸對酚亞硫酸基轉移酵素的影響

Abstract

酚亞硫酸基轉移酵素 (Phenol sulfotransferase) 催化轉移PAPS上的亞硫酸基至酚類、醇類和胺類等化合物。酚亞硫酸基轉移酵素被認為是一種具解毒功能的酵素，它有寬廣的受質特異性及對親脂性化合物具高親合力。氧化還原可以控制酚亞硫酸基轉移酵素的活性。氧化態的酚亞硫酸基轉移酵素比還原態的酚亞硫酸基轉移酵素多10倍的比活性。過去的研究證明C66S會隨時間失去轉移和生理兩種反應的活性。根據親合力的標定、定點突變、亞硫酸基轉移酵素相關的酵素結晶和電腦分子模擬等結果。我們認為胺基酸63-68這段環狀區會影響PAP的結合，這段具彈性的環狀區像是一個蓋子，這蓋子是重要的且關係核甘酸的釋放。當酵素氧化時，雙硫鍵形成而造成蓋子打開。在酚亞硫酸基轉移酵素中有五個半胱胺酸，從電腦模擬中，我們認為Cys66、Cys82和Cys232可能會相互作用。為了探討半胱胺酸的影響，我們利用定點突變的技術產生4個突變株，分別是C66SC283SC289S、C82SC283SC289S、C232SC283SC289S和C82SC232S，這四個突變株被設計來證明模擬的假設和偵測形成雙硫鍵的半胱胺酸。在這研究中我們感興趣的是確認氧化後造成蓋子打開、生理反應活性提高的雙硫鍵是由哪兩個半胱胺酸所造成。從我們初步得到的實驗結果，在氧化還原的環境中可能會形成雙硫建的是Cys66-Cys232和Cys82-Cys232。

Phenol sulfotransferase (PST) catalyzes the transfer of sulfuryl group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to phenols, hydroxylamines, alcohols, and amines (3,4,5). PST is known as an enzyme of detoxication with broad specificity and high affinity for lipophilic compounds (4,8). Redox may control the activity of phenol sulfotransferase (15). The activity of oxidized form of the PST, which shows a 10-fold increase in specific activity over the reduced form (11). The previous demonstration that C66S only a gradual time-dependant decline in both physiological and transfer activities (11). According to a previous study by affinity labeling (14), site-directed mutagenesis (11), the crystal structure of related sulfotransferase (17-22) and molecule modeling of PST (chen and yang). It is interesting that the fragment of PST, from residues 63 to 68, affect PAP binding. We propose the flexible loop be like a cover. The cover is important and affects the releasing of nucleotide. When the enzyme in oxidation condition disulfide bond form, and the cover is open. There are five cysteines in a PST polypetide chain. Model of PST by computer, we infer that the position Cys66, Cys82, and Cys232 of PST are mutually nearby. To study the effect of cysteines, site-directed mutagenesis is used to produce four mutants, they are C232SC283SC289S, C82SC283SC289S, C66SC283SC289S, and C82SC232S. Four cysteines mutants are designed to verify this proposed model and to identify the cysteines that involve in the formation of disulfide bond. Our primary interest in this study is to identify which cysteines may be responsible for the effect of oxidation. Our results suggest that redox environment involves oxidation at Cys66, a process shown to occur by formation of a mixed disulfide with Cys232 or Cys82.