酪胺酸亞硫酸基轉移酶之非典型催化動力學-受質抑制作用及雙相動力學

Abstract

酪胺酸亞硫酸基轉移酶(tyrosylprotein sulfotransferase, TPST)是屬於膜上亞硫酸基轉移酶的一種，在胜肽或蛋白質的特定酪胺酸上修飾亞硫酸基團，改變胜肽或蛋白質物理化學特性，包含親疏水性、表面電位與結構折疊，進而導致許多生理現象的發生，目前已經發現蛋白質亞硫酸化在發炎反應、凝血反應與病毒入侵等都生理現象上扮演重要的功能。雖然TPST在蛋白質後修飾佔有極重要的功能，目前卻仍甚少有相關於酪胺酸亞硫酸基轉移酶的催化機制的研究。我們的實驗發現TPST活性對亞硫酸根受體與供體濃度的催化曲線是不符合一般常見的米氏動力學，在受體濃度的飽和曲線中展現了雙相動力學，在供體濃度的飽和曲線中展現了雙相動力學與受質抑制的現象，並且在不同供體濃度下受體濃度的飽和曲線會展現不同的動力學曲線。這些動力學現象表示TPST具有特殊的催化調控。而相似的動力學現象在細胞質亞硫酸基轉移酶中也有被觀察到，並且已經有相當多的研究，而酪胺酸亞硫酸基轉移酶與細胞質亞硫酸基轉移酶在三級結構上是非常相似的。我們分析所觀察到的動力學現象並參考過去細胞質亞硫酸基轉移酶的催化機制提出TPST的催化模型：TPST屬於二聚化酵素，酵素在受體與供體結合上都是屬於負協同結合，並且存在Homotropic活化導致雙相動力學。供體存在錯誤的結合方式，錯誤的蛋白質複合物將沒有受體結合能力。當二聚體中的其中一單體失去受體結合能力時，受體的異位調節的功能將不會發生。當二聚體的兩個單體都無法結合受體時，酵素將會失去活性。

Tyrosylprotein sulfotransferase (TPST) is a membrane associated sulfotransferase that catalyzes the protein tyrosyl sulfation (PTS), which transforms a sulfonate group to specific tyrosine residue. PTS can change the chemical characteristic of a protein including surface charge, structure and hydropathic that in turn affects many biological phenomena. It has been established that PTS influence many physiological mechanisms, including inflammatory response, blood coagulation and entrance of the virus. However, little studies have been devoted on the catalytic mechanisms of TPST. Our studies on the catalysis of TPST indicate an atypical kinetics profile. The plots of initial velocity versus sulfonate acceptor and sulfonate donor concentrations give biphasic kinetic phenomenon for both saturation curve of sulfonate acceptor and sulfonate donor, inhibition of sulfonate donor under high sulfonate donor concentration. These phenomena show catalytic mechanism of TPST possesses distinct catalytic regulation. Such atypical kinetics have been reported with cytosolic sulfotransferase and is firstly observed in this study. The core structure of TPST is very similar to that of cytosolic sulfotransferase. Catalytic mechanisms of TPST are proposed based on kinetics results and are compared to those of cytosolic sulfotransferase. We propose that, as a dimeric enzyme, TPST sulfonate acceptor and sulfonate donor binding are negatively cooperative, and homotropic activation results in biphasic kinetics. There are wrong binding mode on sulfonate donor binding. The wrong sulfonate donor-enzyme complex is unable to associate sulfonate acceptor. When one subunit of dimeric TPST form the wrong complex and lose sulfonate acceptor binding ability, homotropic effect of substrate would not occur. Even, when two subunits of dimeric TPST form the wrong complex, catalytic activity would lose. Sulfonate donor induced inhibition can be observed.