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-C-14]malonate into C-20 to C-24 VLCFAs Of etiolated leek seedlings was strongly inhibited by the chloroacetamide metazachlor (I-50 approximate to 10-100 nM). The I-50 values of inhibition of incorporation into each VLCFA decreased with their chain length [I-50(20:0) approximate to 1 mu M, I-50(22:0) approximate to 100 nM I-50(24:0) approximate to 10 nM]. With a microsomal preparation from leek seedlings an in vitro elongase assay to quantify chloroacetamide activity was developed using [2-C-14]malonyl-CoA and acyl-CoA (16:0-CoA to 22:0-CoA) as substrates. Under our assay conditions the I-50 values of inhibition by metazachlor of each step of acyl-CoA elongation were determined to be 0.5 mu M (for elongation of 16:0-CoA), 1.7 mu M (18:0-CoA), 0.7 mu M (20:0-CoA), and 0.5 mu M (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 ic) the tight binding of the inhibitor to the target enzyme. (C) 2000 Academic Press.