Kinetic studies on the inhibition of acetolactate synthase by pyrimidinylsalicylic acids

The inhibition of acetolactate synthase (ALS) from etiolated pea seedlings by 2-(4, 6-dimethoxypyrimidine-2-yloxy) benzoic acid (compound 2), one of the pyrimidinylsalicylic acid (PS) compounds, was desensitized by SH inhibitors but not at the alkaline pH. PS compounds including pyrithiobac (2-chloro-6-(4, 6-dimethoxypyrimidine-2-ylthio)benzoic acid) tested in this study inhibited ALS activity in the mixed-type manner with respect to pyruvate and in the non-competitive with respect to thiamine pyrophosphate (TPP). These compounds inhibited both of the two molecular species of ALS which were partially purified from pea seedlings and exhibited slow-binding properties to the crude preparation of ALS from pea seedlings in the extended-time-course experiments. The inhibition constants of the initial inhibition by PS compounds were 13- to 26-fold larger than those of the final steady state and the maximal first order rate constant (0.69min-1) for transition from the initial to the final steady state of the inhibition of pyrithiobac was nearly identical to those reported on other widely acknowledged ALS inhibitors. From these results, we conclude: PS compounds are slow-binding inhibitors to ALS activity, categorized in the sulfonylurea type. The binding site of PS compounds on the enzyme is considered to be located on the similar site to sulfonylureas. This binding site is judged not to be located on the regulatory center(s) but on the allosteric site in a wide sense near the catalytic center, which overlaps partially with the binding site of pyruvate and does not overlap with the binding site of TPP.