[106] Isopropylmalate isomerase (Neurospora)☆☆☆
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Publisher SummaryThis chapter describes the assay, purification, and properties of isopropylmalate isomerase. It catalyzes the interconversion of α- and β-isopropylmalate. This reaction is involved in the biosynthesis of leucine in Neurospora, yeast, and Enterobacteriacae. The involvement of the unsaturated compound––dimethylcitraconate (DMC)––as a true intermediate has not been established, but its production in the in vitro reaction and its interconversion to the two isopropylmalate isomers serves as the basis of the assay procedure. Of the three compounds involved in the reaction, only DMC, by virtue of its conjugated double bonds, absorbs ultraviolet light in the region of 210–250 mμ. As a consequence, the reversible conversion of α- and β-isopropylmalate to DMC can be easily measured by following the rate of increase in absorbance of a reaction mixture at 235 mμ. Because the equilibrium and the rate of the reaction favor the production of DMC from β-isopropylmalate, β-isopropylmalate is the preferred substrate for enzyme assay. Synthetic isopropylmalates and dimethylmesaconate are effective inhibitors of the enzyme. The enzyme is inhibited by Hg2+ and Cu2+ at 10-4 M, and preincubation with the N-ethylmaleimide yields 65% inhibition at 10-3 M.

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