Lipoamide Dehydrogenase from Baker’s Yeast. Improved Purification and Some Molecular, Kinetic, and Immunochemical Properties

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Abstract
The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker''s yeast .apprx. 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (.apprx. twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme possessed diaphorase activity catalyzing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki [inhibition constant] 31 .mu.M). The native enzyme (MW 117,000) was composed of 2 apparently identical subunits (MW 56,000), each containing 0.96 FAD residues and 1 cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val and Cys. The lipoamide dehydrogenases of baker''s and brewer''s yeast were immunologically identical, but no cross-reaction with mammalian lipoamide dehydrogenases was found.