Cadmium(II)-stimulated enzyme activation of Arabidopsis thaliana phytochelatin synthase 1

Phytochelatin (PC) a class of heavy metal binding peptides is synthesized from the tripeptide glutathione (GSH) and/or previously synthesized PC in a reaction mediated by PC synthase (PCS) In the present study the PC production rate catalyzed by recombinant Arabulopsis PCS1 (rAtPCS1) in the presence of a constant free Cd (II) level increased steadily and the kinetic parameters were approximated using a substituted-enzyme mechanism in which GSH and bis(glutathionato)cadmium acted as co-substrates In contrast the PC production rate as a function of GSH concentration at a constant total Cd(II) concentration reached a maximum which shifted toward higher GSH concentrations as the concentration of Cd(II) was increased These observations are consistent with the suggestion that rAtPCS1 possesses a Cd(II) binding site where Cd (II) binds to activate the enzyme The affinity constant optimized using a one-site mathematical model successfully simulated the experimental data for the assay system using lower concentrations of Cd(II) (5 or 10 mu M) but not for the assay using higher concentrations (50 or 500 mu M) where a sigmoidal increase in PCS activity was evident Furthermore the PCS activity determined at a constant GSH concentration as a function of Cd(II) concentration also reached a maximum These findings demonstrate that rAtPCS1 also possesses a second Cd(II) binding site where Cd(II) binds to induce an inhibitory effect A two-site mathematical model was applied successfully to account for the observed phenomena supporting the suggestion that rAtPCS1 possesses two Cd(II) binding sites (C) 2010 Elsevier Inc All rights reserved