Ketol-acid reductoisomerase from barley (Hordeum vulgare): Purification, properties, and specific inhibition

Jörg Durner, Oliver C. Knörzer, Peter Böger

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29 Scopus citations

Abstract

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 μmol 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 Km values of the purified KARI for acetolactate, acetohydroxybutyrate, and NADPH (determined with acetohydroxybutyrate) were 11, 38, and 4.3 μM, respectively. The Vmax obtained with acetohydroxybutyrate was 1.8 μmol min-1 mg-1; the corresponding value for acetolactate was 0.16 μmol 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 μM (acetohydroxybutyrate), respectively.

Original languageEnglish
Pages (from-to)903-910
Number of pages8
JournalPlant Physiology
Volume103
Issue number3
StatePublished - Nov 1993
Externally publishedYes

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