硫胱胺基酸氧化與酚亞硫酸基轉移酵素穩定性關係之探討

Abstract

酚亞硫酸基轉移酵素是一個解毒酵素，在生物體內主要負責亞硫酸基 的轉移，具有相當重要的功能。此酵素內含五個cysteine，這些cysteine 在酵素中所扮演的角色目前並不清楚。我們利用各種物理和化學方式對野 生株及cysteine突變株進行研究，希望能了解當單一個cysteine突變為 serine時，對酵素的活性是否有影響，並且希望能找出使酵素更穩定的方 法，以利未來進行此與酵素有關之研究。實驗結果發現：(1)在氧化還原 實驗中，將酚亞硫酸基轉移酵素以氧化劑處理，野生株及突變株之酵素同 樣會受氧化劑影響，而且在酵素氧化的過程中可以明顯觀察到：酚亞硫酸 基轉移酵素的b型酵素會先改變活性，a型酵素的活性則維持一段時間才變 化；以GSH及DTT兩種不同還原劑去處理野生株以及突變株，發現還原劑對 不同酵素的影響相同，且不同還原劑對酵素活性會有不同的影響。(2)在 酵素穩定性實驗中，以nucleotides、 glycerol、EDTA、sucrose保護野 生株及突變株酵素，發現這些試劑都能穩定酵素，而且將酵素放置在含 有100 mM Bis-Tris propane (pH 7.0)，1 mM DTT，1 mM EDTA，10 % (v/v) glycerol , 125 mM sucrose , 0.5 mM adenosine的4℃環境中， 會使酵素活性維持三星期以上。 Phenol sulfotransferase, a member of the enzyme of detoxification, plays a very important role in biological system. There are five cysteine residues in phenol sulfotransferase, but their function is not clear. To understand the effects of cysteine on the enzyme activity, we used various physical or chemical methods to treat wild type and cysteine mutants of phenol sulfotransferase. The results indicate the following. (1) Oxidation of wild type and mutants by GSSG affecttheir enzymatic activity. During the process of oxidation, we observedthat the b-form phenol sulfotransferase responded quickly with changes in the activity profile upon incubation with oxidation. The activityof the a-form enzyme was changed after a longer incubation period. However incubation with different reducing reagents, these enzymes have different effects. (2) We used a variety of compounds, such as nucleotides, glycerol, EDTA and sucrose, to stabilize the wild type and mutant phenol sulfotransferase. These regents protect these enzymes from oxidation. We found that the enzyme is stable for at least three weeks under the incubation conduction : 100 mM Bis-tris-propane at pH 7.0, 1 mM EDTA, 10% (v/v) glycerol, 125 mM sucrose, 0.5 mM adenosine at 4℃.