Physical and kinetic properties of acetohydroxyacid synthase from wheat leaves

Abstract

L. 1996. Physical and kinetic properties of acetohydroxyacid synthase from wheat leaves. -Physiol. Plant. 98: 824-832.Acetohydroxyacid synthase (AHAS, EC 4.1.3.18; also known as acetolactate synthase), which" catalyses the first reaction common to the biosynthesis of the branchedchain amino acids' L-valiiie, L-leucinc and L-isoleucine. and is the target of several classes of herbicides, has been studied m hydroponically-grown seedlings of wheat (Trincum aestivum L. cv. Vulcan). Enzyme activity was greater in leaves than roots, reaching a maximum between 4 and 6 days after germination. AHAS was associated with the chloroplasts after centrifugation in a density gradient. A preparation of the enzyme was obtained from wheat leaves which gave a single band after electrophoresis in native gels but was resolved by denaturing sodium dodecyl sulphatc-polyaciylamide ael electrophoresis into three polypeptide bands of molecular mass 58, 57 and 15 kDa. the native molecular mass was approximately 128 kDa. AHAS had optimum activity at pH 7 and did not require the addition of flavin adenine dinucleotide (F.AD), thiamine pjTophosphate (TPP) and MgCU for activity. The enzyme did nol display typical hyperbolic kinetics, in that the double reciprocal plot of activity against pyruvate concentration was non-linear The concentration of pyruvate that gave half of the maximum activity was 4 niM. Sulfonylurea and imidazolinone herbicides were potent inhibitors of wheat leaf AHAS, with 50% inhibition being observed at concentrations of 0.6 and 0.3 piW for chlorsulfuron and metsulfuron methyl, respectively, and at 2.5, 5 and 10 ]iM for imazaqum, imazethapyr and imazapyr. Inhibition by both classes of compounds was reversed by removal of the inhibitor. Progress curves of product formation against time in the presence of the herbicides were non-linear, and based on the assumption that inhibition by the sulfonylureas was of the slow, tight-binding type, estimates of 0.17 and 0.1 nM were obtained for the dissociation constants ol chlorsulfuron and metsulfuron methyl, respectively, from the steady-state enzyme-inhibitor complex.

Ion Chromatographic Analysis of Selected Free Amino Acids and Cations To Investigate the Change of Nitrogen Metabolism by Herbicide Stress in Soybean (Glycine max)

“…Acetohydroxyacid synthase, also known as acetolactate synthase (ALS; EC 4.1.3.18), catalyzes the first reaction common to the biosynthesis of the branchedchain amino acids L-valine, L-leucine, and L-isoleucine and is the target of the sulfonylurea herbicide group (Southan and Copeland, 1996;Hattori et al, 1995). Since the late 1980s, these herbicides have been studied and applied in agriculture.…”

Expression, Purification, Characterization, and Reconstitution of the Large and Small Subunits of Yeast Acetohydroxyacid Synthase

“…Rather, its existence is deduced from the presence of an open reading frame (ORF) immediately 3′ to, and sometimes overlapping, that for the large subunit. Due to the low abundance and high lability of the eucaryotic AHAS, purification of the enzyme from its native source is difficult and results in low yield (43)(44)(45). With the availability of the cloned genes (corresponding to the large subunit of bacterial AHAS), studies on the eucaryotic AHAS have been carried out with recombinant enzymes expressed in E. coli (46)(47)(48)(49).…”