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

The inhibition of very-long-chain fatty acid (VLCFA) synthesis by chloroacetamide herbicides was studied in vivo as well as in vitro. Incorporation of [2-14C]malonate into C20 to C24 VLCFAs of etiolated leek seedlings was strongly inhibited by the chloroacetamide metazachlor (I50 ≈ 10–100 nM). The I50 values of inhibition of incorporation into each VLCFA decreased with their chain length [I50(20:0) ≈ 1 μM, I50(22:0) ≈ 100 nM, I50(24:0) ≈ 10 nM]. With a microsomal preparation from leek seedlings an in vitro elongase assay to quantify chloroacetamide activity was developed using [2-14C]malonyl-CoA and acyl-CoA (16:0-CoA to 22:0-CoA) as substrates. Under our assay conditions the I50 values of inhibition by metazachlor of each step of acyl-CoA elongation were determined to be 0.5 μM (for elongation of 16:0-CoA), 1.7 μM (18:0-CoA), 0.7 μM (20:0-CoA), and 0.5 μM (22:0-CoA). Furthermore, it could be shown that inhibition increased with decreasing concentrations of acyl-CoA primer substrate. Using the chiral chloroacetamide metolachlor acyl-CoA elongation was only inhibited with the herbicidally active S-enantiomer while the R-enantiomer had no influence. As demonstrated by 20:0-CoA elongation the I50 value decreased about 10-fold after preincubation of the enzyme preparation at higher temperatures. Furthermore, enzyme activity could not be recovered by dilution of the enzyme–inhibitor complex. These findings strongly support the hypothesis that a covalent binding of metazachlor to its target may occur. From this study the mode of action of herbicidal chloroacetamides is disclosed as a strong inhibition of VLCFA synthesis resulting from (a) an inhibition of each elongation step in series, (b) the increase of that inhibition with the decrease of acyl-CoA primer substrate concentration, and (c) the tight binding of the inhibitor to the target enzyme.