Divalent Cation Inhibition of 3-Ketodisaccharide Synthesis by Agrobacterium tumefaciens

Zinc and copper ions at 1.0 mM effectively inhibited synthesis of 3-ketodisaccharides (3-KD) by washed cells of Agrobacterium tumefaciens. Cu2+ at 0.1 mM completely inactivated cell·free preparations of the synthetic enzyme, hexopyranoside: cytochrome c oxidoreduc· tase. This ion also inhibited uptake of disaccharides. Since Zn2+ blocked disaccharide uptake but had no effect on the synthetic enzyme, it apparently inhibited 3-KD synthesis primarily by preventing the sugar from reaching the enzyme. Zn2+ inhibition of 3-KD synthesis and disaccharide uptake was pronounced at pH 7.5 and weak at pH 6.0. The effect of Zn2+ was completely reversed by 10.0 mM Mg2+ at pH 6.0. Zn2+ inhibition was reversed by 10.0 mM ethylenediaminetetraacetic acid (EDTA) at pH 6.0 or 7.5. Sensitivity to external pH, as well as reversibility by Mg2+ and EDTA, suggested that the site of Zn2+ inhibition of disaccharide transport was on or near the cell surface. Cells preloaded with Zn2+ also displayed inhibition of 3-KD synthesis as well as disaccharide uptake. The inhibition was intensified by prolongation of preloading time or by increased concentrations of Zn2+. The inhibition was strong in cells preloaded with Zn2+ at pH 7.5 but weak in those pretreated similarly at pH 6.0. Inhibition in cells treated with Zn2+ at pH 6.0 was completely reversed by post·treatment with 10.0 mM Mg2+. Partial alleviation occurred when cells treated with Zn2+ at pH 7.5 were post·treated with Mg2+, These data suggested that the effect of Zn2+ resulted from a rather stable binding of the ion to structures or sites required for sugar uptake. Noncompetitive inhibition of disaccharide uptake by Zn2+ suggested that the ion interacted with cells at points other than the sugar binding sites required for transport.