Function and structure of peculiar yeast alcohol dehydrogenases of which activities are reversibly inhibited by Zn^<2+>

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We found that a methanol utilizing yeast, Candida sp. N-16, had two types of NAD-dependent alcohol dehydrogenases. One was a constitutive enzyme (ADH-MI) and the other and inducible enzyme (ADH-MII). These enzyme were inhibited by Zn^<2+>.The purified preparation of ADH-MII was examined in the presence of Zn^<2+> by circular dichroism (CD) analyze. The CD spectrum of ADH-MII was different from those of ADHs from horse liver and baker's yeast. These spectral data indicate that the conformation of ADH-MII changed in the presence of Zn^<2+>. Besides, 20 N-terminal amino acids of ADH-MII were determined.ADH-MI was extremely unstable, but remained stable in the presence of ammonium sulfate (about 800 mM). The enzyme was purified in the presence of ammonium sulfateas an electrophoretically homogeneous protein from cells grown on ethanol. The molecular weight of the native enzyme was estimated to be about 120,000. It consisted of two subunits (90,000 and 40,000). The Km values for ethanol and NAD were 1.25 x 10^<-3> M and 6.25 x 10^<-5> M, respectively. The Ki value for Zn^<2+> was 5.35 x 10^<-5> M.Alcohol dehydrogenase activities in cell-free extracts prepared from other methanol-utilizing yeasts, Hansenula polymorpha and Pichia pastoris, were also inhibited by a low concentration of Zn^<2+>.