[99] α-Hydroxy-β-keto acid reductoisomerase (Neurospora crassa)☆

Publisher Summary This chapter discusses the assay, purification, and properties of α -hydroxy- β -keto acid reductoisomerase. The reactions catalyzed by this enzyme results in the formation of α , β -dihydroxy acid precursors of valine and isoleucine, respectively. The enzyme from all sources shows similar properties—namely, a requirement for Mg 2+ and nicotinamide adenine dinucleotide phosphate (NADPH) and a pH optimum in the basic range. The reaction is followed by measuring the oxidation of NADPH spectrophotometrically at a wavelength of 340 m μ . The optimal pH for the enzyme is 7.5 for both substrates α -aceto- α -hydroxybutyrate and α -acetolactate. The same pH optimum is found for the reductoisomerase from Neurospora , E. coli , and Salmonella. Purified reductoisomerase from Neurospora is very unstable in the absence of cofactors. It is relatively stable when kept at pH 7.5 in the presence of 10 -2 M MgSO 4 , 10 -3 M 2-mercaptoethanol, and 10 -4 M NADPH in Tris buffer. The activity of the enzyme stored at -10° declines 70–80% in five days. Electrophoresis on starch gel shows two protein bands, but only one of these is active in the oxidation of NADPH in the presence of the substrates α -acetolactate and α -aceto- α -hydroxybutyrate.