KETOL-ACID REDUCTOISOMERASE FROM BARLEY (HORDEUM-VULGARE) - PURIFICATION, PROPERTIES, AND SPECIFIC-INHIBITION

Ketol-acid reductoisomerase (KARI, EC 1.1.1.86) was purified to homogeneity from etiolated barley shoots (Hordeum vulgare) using anion exchange, Red-Sepharose, hydrophobic interaction, and chromatofocusing steps. Purification yielded 0.25 to 0.27 mg of pure KARI per 100 g fresh weight of starting material. The specific activity of the purified enzyme was 6 mu mol of NADPH oxidized min(-1) mg(-1) with acetohydroxybutyrate as substrate. The native enzyme had an apparent molecular weight of 115,000 as estimated by gel filtration and appeared to be a homodimer with a subunit molecular weight of 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The K-m values of the purified KARI for acetolactate, acetohydroxybutyrate, and NADPH (determined with acetohydroxybutyrate) were 11, 38, and 4.3 mu M, respectively. The V-max obtained with acetohydroxybutyrate was 1.8 mu mol min(-1) mg(-1); the corresponding value for acetolactate was 0.16 mu mol min(-1) mg(-1). The enzyme showed optimum activity at pH 7.5. When either acetolactate or acetohydroxybutyrate was used as substrate, the experimental herbicidal compound 2-dimethyl-phosphinoyl-2-hydroxyacetic acid inhibited the purified KARI in a time-dependent and reversible manner. The initial inhibition was strictly competitive. The inhibition constant values were 0.46 (using acetolactate as substrate) and 0.19 mu M (acetohydroxybutyrate), respectively.