蛋白質亞硫酸基轉移酶及3’磷酸腺甘酸5’磷酸硫酸合成酶之螢光檢測法

Abstract

在許多藥物以及異體化合物的生物轉化中，亞硫酸化是一個相當重要的反應路徑，亞硫酸轉移酵素(sulfotransferase, ST)利用3’硫酸腺甘酸 5’磷酸硫酸(3’-phosphoadenosine 5’-phosphosulfate, PAPS)當作亞硫酸基的來源，進行以上所述反應的催化，而PAPS則是由無機硫以及三磷酸腺甘(ATP)經由3’硫酸腺甘酸 5’磷酸硫酸合成酶(3’-phosphoadenosine 5’-phosphosulfate synthetase, PAPSS)催化而成。蛋白質亞硫酸化的現象已經被證實了五十多年，酪胺酸亞硫酸化為一種普遍存在於所有的多細胞生物中的後修飾作用。最近幾年，這種蛋白質亞硫酸化的現象也被發現會發生在絲胺酸和蘇胺酸上，然而，催化這個反應的酵素尚未被發現。本論文主要分為兩部份，第一部份主要探討的是 PAPSS螢光檢測法，此活性檢測法的操作結合了酚亞硫酸轉移酵素(phenol sulfotransferase, PST)的生理活性，為一靈敏度極高的活性檢測法。第二部份則是探討蛋白質亞硫酸轉移酵素的活性檢測方法，檢測系統的主旨是利用重組的PST催化受質4-methylumbelliferyl sulfate(MUS)做為亞硫酸基提供者並轉移至3’-phosphoadenosine 5’-phosphate (PAP)再生PAPS ，此核苷酸的亞硫酸基再由蛋白質亞硫酸轉移酵素催化，反應後所產生的4-methylumbelliferone (MU)之螢光變化可用來定量蛋白質亞硫酸轉移酵素的活性。

Sulfation is a major pathway in the biotransformation of many drugs and other xenobiotic compounds. The sulfotransferase (ST) enzymes that catalyze these reactions use 3*-phosphoadenosine 5*-phosphosulfate (PAPS) as a sulfate donor co-substrate. The synthesis of PAPS from inorganic sulfate and ATP is catalyzed by PAPS synthetase (PAPSS). Protein sulfation has been known for 50 years. Tyrosine O-sulfation is a common post-translational modification for all multicellular organisms. It was recently discovered that there are similar phenomena in serine and threonine. However, the enzyme which catalyzes this transformation and its mechanism are still unknown. In the first part of this thesis, a continuously flouremetric assay for PAPS synthetase was developed for the first time. It was performed by coupling with the physiological reaction of phenol sulfotransferase (PST). In the second part of the thesis, we developed a very effective assay for the sulfotransferase that catalyzes the sulfation of serine and threonine. It utilized PAPS regenerated from 3-phosphoadenosine 5-phosphate (PAP) by a recombinant PST using 4-methylumbelliferyl sulfate (MUS) as the sulfuryl group donor. The change in the fluorescence intensity of 4-methylumbelliferone (MU) corresponded directly to the amount of active protein sulfotransferase.