標題: 探討細胞質亞硫酸基轉移酵素之受質篩選機制
The substrate-selecting mechanism of cytosolic sulfotransferase 1 and cytosolic sulfotransferase 2
作者: 李雨澄
Li, Yu-cheng
楊裕雄
Yang, Yuh-Shyong
生物科技學系
關鍵字: 亞硫酸基轉移酵素;cytosolic sulfotransferase
公開日期: 2011
摘要: 細胞質亞硫酸基轉移酵素可催化小分子之亞硫酸基轉移,例如藥物、荷爾蒙、膽酸、神經傳導物質等。依據催化物質之分類主要可分為兩大類,一類為催化苯酚類物質之酵素(也稱為亞硫酸基轉移酵素1),另一類為催化脫氫表雄甾酮類物質之酵素(也稱為亞硫酸基轉移酵素2)。亞硫酸基轉移酵素1催化小分子苯酚類物質之硫基轉移,像對硝基本酚;而亞硫酸基轉移酵素2催化脫氫表雄甾酮(酲)類物質之硫基轉移像脫氫表雄甾酮類。這類酵素皆可容納不同之受質,但這兩類酵素如何篩選它們之受質尚未研究清楚。 經由結構與序列之分析指出,在亞硫酸基轉移酵素1與2活性區當中有很大之差異,我們提出在亞硫酸基轉移酵素1活性區中81號胺基酸(苯丙氨酸)扮演重要之角色於分辨苯酚類與酲類受質。一系列對於81號胺基酸之突變顯示F81Y、F81M仍保留酵素對於苯酚類之活性。F81A、F81G、F81L、F81W之突變顯示降低了酵素對於苯酚類之活性,而增加了酵素對於酲類之活性。在亞硫酸基轉移酵素2中與81號胺基酸對應之胺基酸,為77號胺基酸色氨酸。然而77號胺基酸之突變W77F, W77Y顯示輕微改變酵素對於酲類之活性,也說明了亞硫酸基轉移酵素2有不同的篩選方式。亞硫酸基轉移酵素2利用雌酮為受質之催化效率(kcat)與亞硫酸基轉移酵素1處於相同層級之催化效率。這結果顯示亞硫酸基轉移酵素2可催化雌酮,說明了亞硫酸基轉移酵素2可催化苯酚類與酲類。 我們可利用實驗結果假設亞硫酸基轉移酵素篩選受質之機制,亞硫酸基轉移酵素1,利用81號胺基酸會造成空間障礙,作為排除含有四面立體結構之酲類。而亞硫酸基轉移酵素2喜愛結構包含多環類之物質,無論是苯酚類或酲類。這樣之機制,符合從硫基提供者轉移硫基至苯酚類或酲類受質上之游離機制。也可說明亞硫酸基轉移酵素1與亞硫酸基轉移酵素2可利用相似之機制轉移硫基,即使這兩類物質之酸度係數(pKa)相距非常大,苯酚類為10而酲類為15,相差了105倍。
Cytosolic sulfotransferases catalyze sulfonation of small molecules such as drug, steroid hormones, bile acids, and neurotransmitters. Two main classes of sulfotransfease were identified according to their selectivity on substrates, which is corresponding to two major gene families among cytosolic sulfotransferases: the phenol SULT family (designated SULT1) and the hydroxysteroid SULT family (designated SULT2). SULT1 catalyzes the sulfonation of small phenolic molecules like p-Nitrophenol. SULT2 catalyzes the sulfonation of alcoholic compounds like dehydroepiandrosterone. These enzymes can adopt broad range of substrates, but how these two classes of sulfotransferases select their own substrates is unclear. Structural and sequential comparisons indicated important variations in enzyme active sites between SULT1 and SULT2. We proposed that an active site amino acid, F81, of SULT1A1 plays important role in distinguishing the phenolic and alcoholic compounds. A series of mutation on this amino acid (F81) showed that F81Y, F81M still maintained SULT activity with phenolic substrates; F81A, F81G, F81L, F81W drastically decreased the SULT activity with phenolic substrates and significantly increased its activity toward alcoholic compounds. The corresponding amino acid for F81 in SULT2A1 was W77. However, W77F, W77Y mutants exhibited little changed activity with alcoholic substrates indicating that SULT2A1 may have different mechanism for selecting substrates. We proposed that the whole substrate binding site of SULT2 was used to select substrate for multi-ring structure. The kcat of SULT2A1 using estrone as substrate was in the same order with SULT1A1. This exhibited the SULT2A1 could catalyze estrone, and it could use either phenolic compounds or hydroxysteroids as substrate. A substrate-selecting mechanism of sulfotransferase was proposed according to our results. SULT1A1 used the side chain of F81 as a spatial barrier for excluding alcoholic substrates that have tetrahedral structures. SULT2 preferred the multi-ring structures as substrates which can be either phenolic or alcoholic compounds. This mechanism is consistent with a dissociative mechanism for the transfer of sulfuryl group from PAPS to alcoholic or phenolic substrates. This mechanism explained how SULT1 and SULT2 may use similar mechanism for sulfuryl group transfer even though the pKa values of hydroxyl group on phenolic (~10) and on alcoholic compound (~15) differ in five orders of magnitude.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079828501
http://hdl.handle.net/11536/47709
Appears in Collections:Thesis