Mechanism(s) of action of heavy metals to investigate the regulation of plastidic glucose-6-phosphate dehydrogenase

Abstract

The regulation of recombinant plastidic glucose-6P dehydrogenase from Populus trichocarpa (PtP2-G6PDH - EC 1.1.1.49) was investigated by exposing wild type and mutagenized isoforms to heavy metals. Nickel and Cadmium caused a marked decrease in PtP2-G6PDH WT activity, suggesting their poisoning effect on plant enzymes; Lead (Pb++) was substantially ineffective. Copper (Cu++) and Zinc (Zn++) exposition resulted in strongest decrease in enzyme activity, thus suggesting a physiological competition with Magnesium, a well-known activator of G6PDH activity. Kinetic analyses confirmed a competitive inhibition by Copper, and a mixed inhibition by (Cd++). Mutagenized enzymes were differently affected by HMs: the reduction of disulfide (C175–C183) exposed the NADP+ binding sites to metals; C145 participates to NADP+ cofactor binding; C194 and C242 are proposed to play a role in the regulation of NADP+/NADPH binding. Copper (and possibly Zinc) is able to occupy competitively Magnesium (Mg++) sites and/or bind to NADP+, resulting in a reduced access of NADP+ sites on the enzyme. Hence, heavy metals could be used to describe specific roles of cysteine residues present in the primary protein sequence; these results are discussed to define the biochemical mechanism(s) of inhibition of plant plastidic G6PDH.