阿拉伯芥植物螯合素合成酶 Tyr 55 突變株重組蛋白之表現與活性分析

Expression and Activity Analysis of Tyr 55 Mutants of Phytochelatin Synthase from Arabidopsis thaliana

10.6342/NTU.2009.01712

眭毓庭

阿拉伯芥 ； 植物螯合素合成酶Tyr 55 ； 突變株 ； Arabidopsis thaliana ； Phytochelatin Synthase ； Tyr 55 ； Mutants

臺灣大學微生物與生化學研究所學位論文

2009年

碩士

莊榮輝

繁體中文

植物螯合素合成酶 (phytochelatin synthase, PCS) 利用 glutathione (GSH) 作為基質合成植物螯合素 (phytochelatin, PC)，以結合入侵植物的重金屬。過去研究指出，在六個不同物種中，PCS 序列在其 Tyr 55 相對胺基酸位置具有半保守性，該位置胺基酸非 Tyr 即 Phe，都含有芳香族基團。實驗發現 AtPCS1 (Y55A) 突變株的活性也下降，代表 Tyr 55 在 PCS 活性中扮演重要角色。在 Tyr 55 可能利用鎘離子作為架橋，將第二個 PCS 帶至基質結合區，以進行催化作用的假說下，我們建構了七種 Tyr 55 突變株。包括五株全長 PCS：AtPCS1 (Y55A), AtPCS1 (Y55D), AtPCS1 (Y55E), AtPCS1 (Y55H) 及 AtPCS1 (Y55F)；與兩株 PCS 的 N-端部份：AtPCS1-N (Y55E) 及 AtPCS1-N (Y55W)。表現之後再進行各種 PCS 蛋白質的活性測定。AtPCS1 (Y55D) 突變株和 AtPCS1 (Y55E) 突變株的活性都下降了，然而若以鎘離子對 GSH 做預處理，它們的活性增加程度比 AtPCS1 (Y55A) 增加程度高出許多。更甚者，以經過 Cd 預處理的 GSH 作為基質，AtPCS1 (Y55H) 的活性不增反減，代表了帶電性質能夠調控 PCS 活性。有趣的是，突變株 AtPCS1-N (Y55W) 沒有出現 PCS 活性，但若將基質 GSH 以 Cd 做預處理，便能回復些微活性，說明了 Trp 可能給活性區帶來立體空間障礙，但是在芳香環陽離子-pi 電子交互作用的幫助下，能回復部分活性。總之，Tyr 55 的芳香帶電性質以及立體空間結構，對 PCS 活性具有重要影響。

Using glutathione (GSH) as the substrate, phytochelatin synthase (PCS, EC 2.3.2.15) catalyzes the synthesis of phytochelatins (PCs) which could bind heavy metals in the plant cell. It was found that the corresponding amino acids of Tyr 55 on the PCS sequences are semi-conserved among six different species. The positions are occupied either by Tyr or Phe with aromatic side chains. In vitro experiments showed that PCS activity decreased in the mutant of AtPCS1 (Y55A) revealing that Tyr 55 is essential for PCS activity. Under the hypothesis that Tyr 55 might play a role in docking the gamma-EC of the second GSH substrate by using Cd as the bridge, site-directed mutagenesis on Tyr 55 has been performed. We constructed five mutants for the full-length AtPCS1: AtPCS1 (Y55A), AtPCS1 (Y55D), AtPCS1 (Y55E), AtPCS1 (Y55H) and AtPCS1 (Y55F); and two mutants for the N-terminal part of PCS (AtPCS1-N): AtPCS1-N (Y55E) and AtPCS1-N (Y55W). After expression of the proteins, the enzymes were analyzed for PCS activity. The activity of the mutants AtPCS1 (Y55D) and AtPCS1 (Y55E) decreased; however, by the addition of Cd with substrate GSH in the pretreatment, AtPCS1 (Y55A) showed higher increase in its activity. Furthermore, the activity of AtPCS1 (Y55H) decreased when pretreated with Cd. These observations indicated that the charge of the substrate might play a role in regulating PCS activity. Interestingly, the mutant AtPCS1-N (Y55W) had no PCS activity; however, by the addition of GSH pretreated with Cd, its activity increased slightly, showing that Trp might provide stereo hindrance near the active site, but the activity could be restored by the support of strong cation-p interaction with Cd by its aromatic ring. In conclusion, the aromatic ring on Tyr 55 might be critical for PCS activity.