INHIBITION OF INORGANIC PYROPHOSPHATASE BY PHOSPHONATE - A SITE OF ACTION IN PHYTOPHTHORA SPP.

The sensitivity of inorganic pyrophosphatase (EC 3.6.1.1) to phosphona te was compared in microorganisms, plants, and mammals. Phosphonate in hibited inorganic pyrophosphatase activity regardless of source, with IC50 values ranging from 39 (human cell lines) to 565 mM (rat liver) w hen assayed at saturating substrate concentration. The ICS, values for phosphonate inhibition of the pyrophosphatases from Phytophthora palm ivora and Phytophthora melonis, plant pathogens susceptible to phospho nate in the field, were 109 and 131 mM, respectively, At a substrate c oncentration close to the K-m value (40 mu M), phosphonate gave an IC5 0 of 34 +/- 15 mM against the pyrophosphatase from P. palmivora, sligh tly lower than the corresponding ICS, value for the pyrophosphatase fr om Saccharomyces cerevisae, 85 +/- 7 mM. Phosphate, the product of pyr ophosphatase and a known allosteric inhibitor of the enzyme in S. cere visae, proved to be a slightly better inhibitor of the P. palmivora py rophosphatase than phosphonate (ICS, of 15 +/- 3 mM). Sensitivity to b oth phosphonate and phosphate was dependent upon the concentration of Mg2+, and increasing concentrations of Mg2+ reduced, but never complet ely abolished, inhibition. Our results suggest that at the levels know n to accumulate within Phytophthora mycelia, phosphonate could inhibit pyrophosphatase activity in vivo. However, since the inorganic pyroph osphatases from yeast and Phytophthora were almost equally sensitive t o phosphonate in vitro, while Phytophthora spp. are much more sensitiv e to phosphonate in vivo than yeast, we conclude that this enzyme is u nlikely to be the site at which phosphonate provides specificity again st Phytophthora spp. when compared to other microorganisms. (C) 1998 A cademic Press.