用分子模擬探討環狀區胺基酸序列64~69對酚亞硫酸基轉移酵素活性的影響

Abstract

本實驗使用類比法 (Homology) 來模擬酚亞硫酸基轉移酵素的蛋白質結構，其骨架則是依據動情激素亞硫酸基轉移酵素(Estrogen sulfotransferase)的X-ray單晶繞射結構(1aqu of PDB )。我們可利用模擬出的酚亞硫酸基轉移酵素結構來解釋突變種酚亞硫酸基轉移酵素(K65ER68G)的獨特活性，並說明胺基酸C66，C82和C232之間的相互關係與功能。由結構中我們發現環狀區胺基酸序列區64~69是位於受質結合區的開口區附近而且可能作為控制受質進出的活動閘門。經過胺基酸序列的分析以及相關已解出結構的亞硫酸基轉移酵素所提供給我們的資訊皆指出此環狀區是屬於易滑動的。由前人所做的實驗數據以及我們所做的模擬結構中，我們可以證明此環狀區域位於酵素活性區的通路上，對於調控產物的釋出是非常重要的。主要的關鍵在於，序列C66 和C82 或是 C66 和 C232 所形成的雙硫鍵能夠固定住此易滑動的環狀區域，並且防止它阻礙到受質的結合或是產物的放出。另外由模擬的結構我們可以發現序列K65與R68的正電荷對於環狀區與酵素的活性區進出口之間的相互關係也是很重要的。這個發現也可以用來解釋為什麼突變種(K65ER68G)酵素只包含b 型(無受質PAP)：此原因為突變後形成的負電荷使得原本可以擋住受質進出口的滑動環狀區遠離，同樣的也可以解釋為何突變種(K65ER68G)酵素的生理反應活性可以不需經由氧化就能達到與野生種酵素氧化後得到的活性一樣。

The structure of phenol sulfotransferase (PST) is build by the method of Homology (Insight II) from the crystal structure of estrogen sulfotransferase (1aqu of PDB). The modeled PST structure is used to explain the unique activity of a mutant (K65ER68G) and to elucidate the function of and interaction among C66, C82 and C232. The loop region 64-69 is found outside of the substrate-binding site and may serve as a door of the entrance or exit of the active site. Data analysis of the amino acid sequence and information from know structure of sulfotransferases all indicate that this region is very flexible. From the previous experimental data and our modeling study, we are able to demonstrate that this region is in the way of the enzyme active site and is important for the regulation of the release of products. Formation of disulfide bonds between C66 and C82 or C66 and C232 immobilized this flexible region and prevent it from hindering substrate binding or product release. The modeled PST structure also shows that positive charges at K65 and R68 are important for the interaction of the loop region with PST active site. This observation explains why only b form (PAP free) of K65ER68G is obtained for the flexible loop region is no longer available as a cover after being mutated to contain a negative charge. The same reason explains the physiological activity of K65ER68G is obtained without oxidation, which is required by wild type PST.