從原核表現人類酪氨酸亞硫酸基轉移酶探討其催化活性與功能

Abstract

酪氨酸亞硫酸基轉移酶負責催化許多分泌性蛋白質和膜上蛋白質之亞硫酸基化。酪氨酸亞硫酸基轉移酶已經在許多哺乳動物和低階的生物，如線蟲和果蠅中被發現，但在酵母菌與原核生物中卻沒有發現到。在大部分的研究中，酪氨酸亞硫酸基轉移酶的來源通常是由天然物與細胞培養方式來製備，為了能夠更加了解酪氨酸亞硫酸基轉移酶的催化特性，我們成功地開發出新的表現系統，從大腸桿菌中純化出人類酪氨酸亞硫酸基轉移酶-2，利用Mass指紋圖譜分析所得到結果確實為人類酪氨酸亞硫酸基轉移酶-2。藉由同位素標定的方式，探討重組的酪氨酸亞硫酸基轉移酶一些基本性質；包括催化是否隨著時間呈線性增加，以及不同的溫度、pH值與金屬離子的濃度對酵素活性之影響，我們也針對此酵素對於polyEAY做酵素動力學試驗。未來我們將使用這一套系統去表現突變的酪氨酸亞硫酸基轉移酶，並尋找負責催化活性的胺基酸與一些尚未解決反應機制的問題。

Tyrosylprotein sulfotransferase (TPST) is responsible for the sulfation of a variety of secretory and membrane proteins. TPSTs have been found in mammalian and metazoans such as Caenorhabditis elegans and Drosophila melanogaster, but not in yeast and prokaryotes. TPSTs have been studied only from natural materials and eukaryotic cells following partial purification. To be able to fully characterize the biochemical properties of TPSTs in depth, we successfully overexpressed and purified human TPST2 in E. coli, and then confirmed by Mass fingerprinting identification. The properties of recombinant TPST2 were characterized using by isotope-based analysis. The optimal temperature, pH profile, and concentration-dependent of MnCl2 for human TPST2 were determined. The kinetic constants of TPST2 using polyEAY as substrate were also determined. Furthermore, the important amino acids responsible for the activity of TPST and its mechanism of actions will be studied.