New Aspects on Inhibition of Plant Acetolactate Synthase by Chlorsulfuron and Imazaquin

Abstract

The sulfonylurea herbicide chlorsulfuron and the imidazolinone herbicide imazaquin were shown to be noncompetitive and uncompetitive inhibitors, respectively, of purified acetolactate synthase from barley (Hordeum vulgare L.) with respect to pyrvuate. From double-reciprocal plots of the time-dependent biphasic inhibition by chlorsulfuron, an initial apparent inhibition constant of 68 nanomolar was calculated (a 0 to 4 minute hssay was used for the initial inhibition), and a final steady-state dissociation constant of 3 nanomolar was estimated. The corresponding constants for imazaquin were 10 and 0.55 micromolar. Specific binding of [14C]chlorsulfuron and [14C]imazaquin to purified acetolactate synthase from barley and partially purified enzyme from corn (Zea mays L.) could be demonstrated by gel filtration and equilibrium dialysis. Evidence is presented that the binding of the inhibitors to the enzyme follows the previously described mechanism of slow reversibility once excess inhibitor has been removed. However, after formation of the slowly reversible complex and subsequent dissociation, both chlorsulfuron and imazaquin seem to permanently inactivate acetolactate synthase. These results add a new feature to the mode of action of these herbicides with respect to their high herbicidal potency. ALS2 (EC 4.1.3.18; also referred to as acetohydroxyacid synthase) catalyzes the first common step in the plant biosynthetic pathway of the essential branched-chain amino acids. For valine and leucine biosynthesis, two molecules of pyruvate are condensed to form 2-acetolactate, while for isoleucine formation one molecule of pyruvate reacts with 2-ketobutyrate in a similar reaction. The enzyme has raised strong interest since structurally unrelated classes of modern and very potent herbicides and inhibitors, the sulfonylureas (16), the imidazolinones (1 8), the triazolo pyrimidines (20)

Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase

“…The inhibition constant for chlorsulphuron was obtained only for the initial phase of the inhibition. The K i (app) of 32n4 nM is similar in magnitude to the value of 68 nM obtained for the barley enzyme [49]. Inhibition of the barley enzyme by chlorsulphuron was shown to be non-competitive, with the slope and intercept of a double-reciprocal plot almost equally affected by the inhibitor.…”

“…do not clearly distinguish between effects on initial rates and rates obtained as an average from a prolonged incubation with inhibitor. Nevertheless, there is broad agreement that the sulphonylureas inhibit in the nanomolar concentration range : barley (chlorsulphuron 33-34 nM [49]), Arabidopsis (chlorsulphuron 2-2n1 nM [20], metsulphuron methyl 80 nM [21]), wheat (chlorsulphuron 600 nM, metsulphuron methyl 300 nM [18]) and several plant species (chlorsulphuron 11-32 nM, metsulphuron methyl 9-46 nM [51]). …”

The oxygenase reaction of acetolactate synthase detected by chemiluminescence

“…As reported previously ALS is irreversibly affected by inhibitors [14,23]. Despite the slow dissociation of the tightly-bound inhibitor from the enzyme a permanent inactivation of the enzyme remains.…”

“…ref. [14]). The sensitive detection of the oxygenase reaction as presented here should allow for further experiments.…”

“…Seeds of barley (Hordeurn vulgare L.) and corn (Zea mays L.) were cultivated as described [5,14]. The purification of ALS from etiolated barley shoots was slightly modified [5].…”