The effect of phosphorylation and Tyr 55 mutation on the catalytic activity of phytochelatin synthase from Arabidopsis thaliana
Date Issued
2011
Date
2011
Author(s)
Lin, Shin-Yu
Abstract
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.
Subjects
phosphorylation
Tyr 55 mutation
phytochelatin synthase
Type
thesis
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