Characterization of phytochelatin synthase from tomato

Abstract

The enzyme that synthesizes Cd‐binding phytochelatins (PCs), PC synthase, has been studied in tomato (Lycopersicon esculentum) cell cultures and plants. This enzyme transfers γ‐GluCys from GSH or PC to either GSH or an existing polymer of (γ‐GluCys)nGly. PC synthase from tomato requires GSH or PCs as substrates but cannot utilise γ‐GluCys or GSSG. PC synthase is activated both in vivo and in vitro by a variety of heavy metal ions, including Cd2+, Ag+, Cu2+, Au+, Zn2+, Fe2+, Hg2+ and Pb2+. In crude protein extracts from tomato cells the enzyme has an apparent Km of 7.7 mM for GSH in the presence of 0.5 mM Cd2+, and exhibits maximum activity at pH 8.0 and 35°C. PC synthase is present in tomato cells grown in the absence of Cd. The level of enzyme activity is regulated during the cell culture cycle, with the highest activity occurring 3 days after subculture. Cadmium‐resistant tomato cells growing in medium containing 6 mM CdCl2 have a 65% increase in PC synthase activity compared to unselected cells. PC synthase is also present in roots and stems of tomato plants, but not in leaves or fruits. The distribution of the enzyme in tomato plants and regulation of PC synthase activity in tomato cells indicate that PC synthase, and PCs, may have additional functions in plant metabolism that are not directly related to the formation of Cd‐PC complexes in response to cadmium.