利用Lys-65及Arg-68定點突變探討酚亞硫酸基轉移酵素核□酸結合位置

Abstract

生物體可以藉由對蛋白質、醣蛋白、醣類、神經傳導物質、荷爾蒙等物質 進行硫酸化，而參與生物體內荷爾蒙調節、訊息分辨、神經傳導與解毒作 用等重要反應。亞硫酸基轉移酵素在亞硫酸化的反應中扮演一重要角色， 它可以從3'-phosphoadenosine 5'-phosphosulfate（PAPS）催化轉移一 亞硫酸基至親核基（nucleophilic）的受質上。此外3'-phospho adenosine 5'-phosphate（PAP）可以為亞硫酸基轉移酵素催化轉移反應 中之輔因子。根據Duffel等人親和性試驗與Jokoby等人對K65GR68E突變株 的研究結果，顯示此區域與核□酸之結合位有密切關係，本研究之目的即 欲以定點突 K65ER68G突變株在比較後發現其對於PAP的Km值增加超 過500倍(6)。若以Adenosine 5'-monophosphate(AMP), Adenosine 2',3'-cycliphosphate 5'-phosphate以及Adenosine 2',5'-bisphosphate取代PAP作為亞硫酸基轉移的輔因子，這些核甘酸在 突變株所顯示的Km、Vmax及Kd與野生株者比較沒有明顯的差異。根據 Yoshimitsu等人所研究的結果，第65個位置上的Lys與第68個位置上的Arg 對於酵素與PAP之間似乎沒有直接關係，但是由K65ER68G突變株所測得Km 值值與Kd值的結果可以看到與野生株有明顯的差異，由此我們發現將 Lys65與Arg68突變後會使酵素產生變化進而改變酵素 Sulfation in biological system plays important roles in regulating structure and function of macromolecules, availability of hormones and neurotransmitters, activation and deactivation of xenobiotics, and elimination of end products of catabolism. Sulfotransferase catalyze all the known biological sulfation which involves the transfer of a sulfuryl group from a common sulfate donor, a nucleotide called 3'-phospho adenosine 5'-phosphosulfate (PAPS). Another nucleotide, 3'-phospho adenosine 5'-phosphate (PAP)A double mutant (K65E+R68G) exhibits over 500-fold increase in Km for PAP with Vmax remained the same （6）. Adenosine 5'-monophosphate (AMP), Adenosine 2', 3'-cycliphosphate 5'-phosphate and Adenosine 2', 5'-bisphosphate were shown to replace PAP as cofactor for sulfuryl group transfer（24）. However neither mutant's Km nor Vmax of these nucleotides differ significantly with that of wild type's. According to the study of Yoshimitsu, et al, Lys 65 and Arg68 were not involved in enzyme PAP binding site. But w