Chang A K, Duggleby R G
Centre for Protein Structure, Function and Engineering, Department of Biochemistry, The University of Queensland, Brisbane QLD 4072, Australia.
Biochem J. 1998 Aug 1;333 ( Pt 3)(Pt 3):765-77. doi: 10.1042/bj3330765.
Acetohydroxyacid synthase (AHAS) catalyses the first step in the synthesis of the branched-chain amino acids and is the target of several classes of herbicides. Four mutants (A122V, W574S, W574L and S653N) of the AHAS gene from Arabidopsis thaliana were constructed, expressed in Escherichia coli, and the enzymes were purified. Each mutant form and wild-type was characterized with respect to its catalytic properties and sensitivity to nine herbicides. Each enzyme had a pH optimum near 7.5. The specific activity varied from 13% (A122V) to 131% (W574L) of the wild-type and the Km for pyruvate of the mutants was similar to the wild-type, except for W574L where it was five-fold higher. The activation by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined. A122V showed reduced affinity for all three cofactors, whereas S653N bound FAD more strongly than wild-type AHAS. Six sulphonylurea herbicides inhibited A122V to a similar degree as the wild-type but S653N showed a somewhat greater reduction in sensitivity to these compounds. In contrast, the W574 mutants were insensitive to these sulphonylureas, with increases in the Kiapp (apparent inhibition constant) of several hundred fold. All four mutants were resistant to three imidazolinone herbicides with decreases in sensitivity ranging from 100-fold to more than 1000-fold.
乙酰羟酸合酶（AHAS）催化支链氨基酸合成的第一步，是几类除草剂的作用靶点。构建了拟南芥AHAS基因的四个突变体（A122V、W574S、W574L和S653N），在大肠杆菌中表达，并对这些酶进行了纯化。对每个突变体形式和野生型的催化特性以及对九种除草剂的敏感性进行了表征。每种酶的最适pH接近7.5。比活性为野生型的13%（A122V）至131%（W574L），除W574L的丙酮酸Km值比野生型高五倍外，突变体的丙酮酸Km值与野生型相似。研究了辅因子（FAD、Mg2+和硫胺二磷酸）的激活作用。A122V对所有三种辅因子的亲和力降低，而S653N与FAD的结合比野生型AHAS更强。六种磺酰脲类除草剂对A122V的抑制程度与野生型相似，但S653N对这些化合物的敏感性有所降低。相比之下，W574突变体对这些磺酰脲类不敏感，表观抑制常数（Kiapp）增加了数百倍。所有四个突变体对三种咪唑啉酮类除草剂均具有抗性，敏感性降低范围从100倍到1000倍以上。
