Inhibition of enzymes of the glycolytic pathway and hexose monophosphate bypass by phosphonate

Previous studies have suggested that the phosphonate: ion (Phi), an isoster e of phosphate, might be a general inhibitor of enzymes which are allosteri cally regulated by phosphate or which have a requirement for divalent catio ns. In this paper, the capacity of Phi to inhibit selected enzymes of this type from Phytophthora palmivora is compared with its effects on the same e nzymes isolated from other organisms, in particular from Saccharomyces cere visiae, which are widely used in linked assays of many enzymes. It was foun d that Phi inhibited the activity of the enzymes from P. palmivora investig ated, though to different degrees. IC50 values (concentration required to i nhibit enzyme activity by 50%) for Phi ranged from 0.74 +/- 0.07 mM (NAD-de pendent glyceraldehyde-3-phosphate dehydrogenase) to 116.1 +/- 7.3 mM (6-ph osphogluconate dehydrogenase). Among the activities tested glucose-6-phosph ate dehydrogenase activity from P. palmivora was significantly more sensiti ve to Phi than the same enzyme from yeast, although its absolute IC50 value (29.0 +/- 3.4 mM) was high in comparison to most fungicides. It was also f ound that the auxiliary enzymes from rabbit muscle (aldolase, glycerophosph ate dehydrogenase, and triosephosphate isomerase) and yeast (glucose-O-phos phate dehydrogenase) used in enzyme-linked assays were all sensitive to Phi , giving IC50 values between 7.7 +/- 0.4 and 73.6 +/- 2.0 mM, a sensitivity comparable to the other enzymes under investigation. Inorganic phosphate a lso inhibited the activity of the enzyme glucose-6-phosphate dehydrogenase and the aldolase/triosephosphate isomerase/glycerophosphate dehydrogenase m ixture with IC50 values of 108.3 +/- 7.7 and 13.0 +/- 0.6 mM, respectively. In conclusion, Phi inhibition was found to be widespread, supporting the h ypothesis that Phi may inhibit several enzymes rather than acting at a sing le unique site. It was also found that the coupling enzymes used in many of the assays for these enzymes were themselves susceptible to Phi and phosph ate inhibition, which must be taken into account in the interpretation of t he results obtained with this type of assay. (C) 2000 Academic Press.