標題: 酚亞硫酸基轉移酵素之光譜研究:酚亞硫酸基轉移酵素受其結合子誘導所產生之光譜變化
Spectroscopic Studies of Phenol Sulfotransferase:Ligand Induced Spectral changes of Phenol Sulfotransferase
作者: 李宗樹
Tzung Shu Li
楊裕雄
Yuh-Shyong Yang
生物科技學系
關鍵字: 酚亞硫酸基轉移酵素;phenol sulfotransferase
公開日期: 2000
摘要: 酚亞硫酸基轉移酵素是以3’-phosphate 5’-phosphosulfate (PAPS)為亞硫酸基提供者,催化亞硫酸基轉移至生物系統中各種的親核受體的反應。雖然許多酚亞硫酸基轉移酵素已被純化,並且已測定過其動力學,但有關其結合子與之結合以及酵素構形變化都還未知。在此研究中,我們使用紫外/可見光吸收光譜、圓二極光譜和螢光光譜等技術,研究酵素受結合子所誘導產生的構形改變,以及結合子環境的改變。我們的實驗數據顯示,經由p-nitrophenol (p-NP)新產生的最大吸收光譜375nm的變化,測定出酵素(二聚體)、adenosine 3’,5’-bisphosphate (PAP)和p-NP的結合比例為1:1:1。此結果暗示著酵素兩活性區的結合能力差別相當大。不同的核甘酸,像是adenosine 5’-monophosphate (AMP)或adenosine 3’-monophosphate (3’-AMP)也都能與酚亞硫酸基轉移酵素緊密的結合。然而它們卻無法誘導出與PAP誘導一樣的光譜改變。以不同的光譜發現,這些核甘酸對酵素構形的影響是不同的。不同的酚類化合物也被用來代替p-NP,觀察它們在酵素與PAP存在下光譜的不同。此結果指出當酚類化合物進入酵素的活性區後,它們會趨向於去質子化的狀態。依據上述的研究,有關酚亞硫酸基轉移酵素所催化的作用機制與抑制現象將會被提出。
Phenol sulfotransferase (PST) catalyzes sulfuryl group transfer from adenosine 3’-phosphate 5’-phosphosulfate (PAPS) to a variety of nucleophilic acceptor in biological system. Although several PSTs have been purified and examined by steady state kinetic methods, little is known about ligands binding and conformational change in these enzymes. In the present study, we investigated conformational change of PST induced by ligands and environmental change of ligands binding site using UV/visible absorption, circular dichroism, and fluorescence techniques. Our experimental data show that the ratio of PST (dimer):adenosine 3’,5’-bisphosphate (PAP):p-nitrophenol (pNP) is 1:1:1, determined through the change of an unique pNP absorption maximum at 375 nm. The result suggests that binding ability of two subunits of PST is significantly different. Different nucleotides, such as adenosine 5’-monophosphate (AMP) or adenosine 3’-monophosphate (3’-AMP), are also tightly bound to PST. Unlike PAP, they do not induce the same spectral change observed with PAP binding. The effects of these nucleotides on the conformational change of PST are indicated by different spectroscopies. Different phenols are used to replace p-NP and, similar to that of p-NP, their spectral differences are observed in the presence of PAP. The result suggests that the phenolic compounds become deprotonated as they get into the active site of PST. Mechanism and inhibition of sulfuryl group transfer catalyzed by PST are proposed base on the above studies.
URI: http://140.113.39.130/cdrfb3/record/nctu/#NT890111026
http://hdl.handle.net/11536/66574
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