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Molecular Characterization And Determination Of Activation Requirements For A Phytochelatin Synthase From Thalassiosira Pseudonana
Phytochelatin synthase (PC synthase) is the enzyme that synthesizes metal binding peptides, called phytochelatins (PCs), from glutathione (GSH). Found in plants and algae, PCs are produced in response to heavy metal exposure. PC production has been studied in many organisms, however PC synthase has only been isolated from a few sources. The most well-studied PC synthase was isolated from Arabidopsis thaliana (AtPCS1). We studied a PC synthase from a marine diatom, Thalassiosira pseudonana. The gene predicted to encode this PC synthase, termed TpPCS3, was cloned and expressed in E. coli and the subsequent enzyme was purified using an immunoaffinity column. As a standard for comparison, AtPCS1 was also purified in the same manner. The results of our enzyme assays showed that both enzymes were susceptible to oxidation following purification, however, TpPCS3 was found to be more sensitive as it became completely oxidized during purification. Non-reducing PAGE analysis showed that oxidation of the TpPC3 and AtPCS1 corresponded with a change in their electrophoretic mobility. This indicates a change in the enzyme's structure which may be attributed to disulfide bond formation. Oxidation resulted in the decrease of AtPCS1 activity and the termination of TpPCS3 activity, suggesting that preservation of reduced thiols is a necessary requirement for PC synthase activation. The optimal activation pH and temperature for TpPCS3 and AtPCS1 were the same, however the two enzymes differed in their kinetic models and their KM values for the substrate Cd(dot)GS2. The KM found for TpPCS3 was an order of magnitude lower than that of AtPCS1. The kinetics of TpPCS3 was best described by a ternary complex mechanism. Finally, we determined that bathocuproine, a specific copper(I) chelator, is capable of reducing the activity of purified TpPCS3 and terminating the activity of purified AtPCS1. Although determination of the metal content of AtPCS1 by ICP-MS did not prove that copper(I) was removed from the enzyme, the data still suggests that metal is necessary for activation as either a substrate or as a structural component. Thus, direct metal binding is required for either enzyme structure or activation.
dissertation or thesis