101] α,β-Dihydroxy acid dehydratase (Neurospora crassa and spinach)

Abstract

Publisher Summary This chapter describes the assay, purification, and properties of α , β -dihydroxy acid dehydratase. The catalysis of dihydroxy acid dehydratase results in the formation of the keto acid analogs of valine and isoleucine. The enzyme is widely distributed in nature, being found in fungi, including yeast, higher green plants, and many different bacteria. α -keto acid produced after incubation of the α , β -dihydroxy acids with the enzyme is converted to dinitrophenylhydrazone, which is solubilized with base and determined colorimetrically. Partial purification of dihydroxy acid dehydratase has been accomplished with Phaseolus radiatus , Spinacea oleracea , Escherichia coli , and Neurospora crassa . For the synthesis of α,β -dihydroxyisovalerate, acetone is first reacted with ethyl chloroacetate in the presence of sodium methoxide. The ethyl DL- α , β -oxidoisovalerate formed is saponified, and the DL-acid resolved by formation of the quinine salts, one of which crystallizes readily to give the optically active natural isomer. The same procedure is used to make α,β -dihydroxy- β -methylvalerate except that methyl ethyl ketone is used as a starting compound instead of acetone.