磷酸化及Tyr 55突變對阿拉伯芥植物螯合素合成酶之催化活性影響

Abstract

阿拉伯芥 (Arabidopsis thaliana) 中含有植物螯合素合成酶 (phytochelatin synthase, PCS, EC 2.3.2.15)，會利用 glutathione (GSH) 為基質來合成植物螯合素 (phytochelatin, PC) 以結合入侵的重金屬，降低對植物的傷害。由於本實驗室以前表現出的PCS重組蛋白質N端帶有容易與金屬螫合的His-Tag，因此本論文試著將重組蛋白質上的Tag以thrombin去除。結果顯示不論是否帶有His-Tag，PCS的活性均無明顯改變。這可能是我們給予的鎘離子濃度和基質GSH超過活性催化所需，所以His-Tag的有無對PCS重組蛋白質來說沒有太大的差異。利用點突變技術將PCS的 N端保守性區域的Tyr 55進行點突變之後，測定不同突變株之表現蛋白質活性，發現同為芳香族的Y55F和Y55W突變株的活性和野生種比起來，僅稍微下降一些，而Y55H和Y55A突變株的活性則劇烈下降。進一步利用酵素動力學計算各突變株的Km和kcat，發現當側基失去芳香基團，酵素的整體催化能力也隨之下降50% 左右。由以上結果我們推測Tyr 55的苯環可以經由cation-pi 作用力，與帶著鎘的GSH複合物結合，以此將第二個基質GSH帶進催化區合成PC。另一方面，本論文以二次元電泳分析在植物體中PCS是否有磷酸化現象，發現未經過鎘逆境處理的阿拉伯芥樣本中，若加入phosphatase會使PCS蛋白質色點位移至pI較大之處，這可能是PCS被去除磷酸化所造成的結果。分別以不同濃度的鎘逆境處理後的阿拉伯芥樣本則無色點位移現象，但是各色點的比例有些不同。推測阿拉伯芥受到鎘逆境時PCS的磷酸化程度會隨之改變，所以二維電泳圖譜上才會呈現出色點比例的差異。分析阿拉伯芥粗抽取液的PCS活性，發現加以phosphatase inhibitor處理會提升PCS的活性，顯示磷酸化的確會對PCS的活性造成影響。本論文也使用免疫沉澱法來純化內生性PCS，將利用質譜儀 (LC-MS/MS) 來分析抗體辨認到的蛋白質身分，以及有無磷酸化修飾的現象。

Phytochelatin synthase (PCS, EC 2.3.2.15) in Arabidopsis thaliana uses glutathione (GSH) as its substrate for the synthesis of phytochelatins (PCs) which could bind to heavy metals to reduce damages to cells. We have expressed AtPCS1 with His-Tag on its N-terminal sequence using E. coli expression system. In this study, His-Tag on AtPCS1 construct was removed by thrombin and the activity was then analyzed. There is no change on PCS catalytic activities whether the recombinant proteins contained His-Tag or not. This might be caused by using high concentration of GSH and Cd in the activity assay solution. To explore possible functions of the putative second substrate binding site, we tested several mutants at Tyr 55. Results showed that the activities of the mutants Y55F and Y55W were slightly lower than that of the wild-type. However, activities of Y55H and Y55A were dramatically decreased. Furthermore, changes on enzyme kinetic parameters of Tyr 55 mutants indicated that the aromatic group on Tyr 55 was important to PCS catalytic activity. These results suggested that Tyr 55 might bind GSH through cation-pi interaction. On the other hand, the two-dimensional electrophoresis (2-DE) analysis revealed that the native PCS in Arabidopsis showed several spots with different pI values. Samples treated with calf intestinal alkaline phosphatase (CIP) showed shifts of PCS spots on 2-DE, which might resulted from dephosphorylation of PCS. Samples treated with various Cd stress were also tested; the pI values of PCS spots were not affected by CIP, but the propotion of spots were slightly changed. These results suggested that phosphorylation of PCS might be enhanced in plants under Cd stress. In addition, PCS activity in Arabidopsis was decreased when sample was treated with CIP, and the activity could be recovered by adding phosphatase inhibitors. The above observations showed that the PCS activity might be regulated by phosphorylation. We also purified the endogenous PCS from Arabidopsis plants by immunoprecipitation and will identify the protein spots and its phosphorylation by LC-MS/MS.