Inhibition of Acyl-CoA Elongation by Chloroacetamide Herbicides in Microsomes from Leek Seedlings

Previous studies have suggested that the phosphonate ion (Phi), an isostere of phosphate, might be a general inhibitor of enzymes which are allosterically regulated by phosphate or which have a requirement for divalent cations. 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 enzymes isolated from other organisms, in particular from Saccharomyces cerevisiae, which are widely used in linked assays of many enzymes. It was found that Phi inhibited the activity of the enzymes from P. palmivora investigated, though to different degrees. IC50 values (concentration required to inhibit enzyme activity by 50%) for Phi ranged from 0.74 ± 0.07 mM (NAD-dependent glyceraldehyde-3-phosphate dehydrogenase) to 116.1 ± 7.3 mM (6-phosphogluconate dehydrogenase). Among the activities tested glucose6-phosphate dehydrogenase activity from P. palmivora was significantly more sensitive 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 found that the auxiliary enzymes from rabbit muscle (aldolase, glycerophosphate dehydrogenase, and triosephosphate isomerase) and yeast (glucose-6-phosphate 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 also inhibited the activity of the enzyme glucose-6-phosphate dehydrogenase and the aldolase/triosephosphate isomerase/glycerophosphate dehydrogenase mixture 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 hypothesis that Phi may inhibit several enzymes rather than acting at a single 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 phosphate inhibition, which must be taken into account in the interpretation of the results obtained with this type of assay.