Biosynthesis of valine and isoleucine in plants II. Dihydroxyacid dehydratase from Phaseolus radiatus

1. 1. An enzyme catalysing the conversion of α,β-dihydroxyisovalerate and α,β-dihydroxy-β-methylvalerate to α-ketoisovalerate and α-keto-β-methylvalerate has been partially purified from green gram (Phaseolus radiatus), and its characteristics studied. 2. 2. A natural inhibitor, heat stable and inorganic in nature, was observed in the crude extracts. 3. 3. The observed Km values for α-β-dihydroxyisovalerate and α,β-dihydroxy-β-methylvalerate were 2.4 · 10-3 M and 9 · 10-4 M, respectively. 4. 4. The enzyme required the presence of a divalent metal ion (Mg2+, Mn2+ or Fe2+) for maximal activity. Heavy metals like Ag+ and Hg2+ were inhibitory. 5. 5. The optimal activity was around pH 8.0 and the optimum temperature at 52°. The activation energy is found to be 12 600 cal/mole. 6. 6. The enzyme was inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and sulphydryl compounds like cysteine, glutathione, 2-mercaptoethanol and 2,3-dimercaptopropanol. The inhibition by p-hydroxymercuribenzoate could not be reversed by any of the sulfhydryl compounds tested.