Cd(II)-Activated Synthesis of Phytochelatins

Abstract

Phytochelatins (PCs), a class of peptides with the general structure (γ-Glu-Cys) _n -Gly (n≥2), are implicated in heavy metal tolerance in higher plants, algae, and a fungal species. Synthesis of the peptides is mediated by an enzyme designated as PC synthase (PCS) from the tripeptide glutathione (GSH). Point mutation of the Arabidopsis PCS demonstrated a catalytic triad of the protein, with the Cys56 thiol group as the acylation site. A peptide elongation reaction proceeds with the acylation of the enzyme by a γ-Gly-Cys moiety of GSH, followed by transfer of the dipeptide to another GSH or previously formed PC. A kinetic analysis of the PC synthesis rate was complicated, as the GSH substrate formed complexes with Cd(II). An assay system in which the free Cd(II) level was kept constant with increasing GSH concentration circumvented this difficulty and demonstrated a substituted enzyme mechanism in which a GSH molecule and a bis(glutathionato) Cd(II) complex acted as co-substrates. The enzyme reaction rate at a constant total Cd(II) concentration showed decreased activity at higher GSH concentrations. This phenomenon was attributable to a reduction in the fraction of Cd(II)-bound enzyme by the decreased free Cd(II) level under higher GSH concentrations, consistent with a mechanism that Cd(II) binding is necessary for the protein to be active. A simulation of the reaction rate demonstrated that the enzyme possesses a Cd(II)-binding site with a dissociation constant of a few tens to one hundred pM. Therefore, PC synthesis is controlled in such a way that the enzyme is activated by the intrusion of Cd(II) ions into cells by binding to the site and the enzyme is deactivated by removing the ion from the activated enzyme through a complexation of the product PCs with Cd(II).