The role of Tyr55 in the active site of Arabidopsis thaliana phytochelatin synthase 1

• Main Authors: Hui-Ming Huang, 黃惠敏
• Other Authors: Rong-Huay Juang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/53035788417730267062

碩士 === 臺灣大學 === 微生物與生化學研究所 === 98 === Phytochelatin (PC) are a group of heavy metal binding peptides in plant cells, which are catalyzed by the enzyme phytochelatin synthase (PCS, EC 2.3.2.15) using glutathione as the substrate. Previous study showed that PCS active site contained six important amino acids contributing to the catalytic activity. Based on the sequence alignment of PCS among several species, Tyr55 was found to be semi-conserved by either Tyr or Phe. In this study, we tried to elucidate the role of AtPCS1 Tyr55 by producing six point mutation recombinants for activity analysis. Our results showed that no activity was detected in Y55D and Y55E, and the activity of Y55A was 9-folds lower as compared to the wild type PCS. According to the cation-p theory, amino acids with aromatic ring such as Tyr and Phe may provide cation-p interactions with Cd ion which might stabilize the second substrate for PCS. Furthermore, recombinant AtPCS1 fused with GFP protein was used for the cellular localization of PCS by Transient expression in onion epidermal cells. The results showed that PCS was localized predominantly in the cytosol. This might be due to the high amount of GSH present in the cell cytosol.