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

我们发现了一株利用甲醇的酵母菌，假丝酵母。N-16有两种依赖NAD的乙醇脱氢酶。一种是构成酶(ADH-MI)，另一种是诱导酶(ADH-MII)。这些酶被锌离子抑制。用圆二色谱(CD)分析了在锌离子存在下纯化的ADH-MII制剂。ADH-MII的CD谱与ADHS的马肝和面包酵母的CD谱不同。这些光谱数据表明，在锌离子的存在下，ADH-MII的构象发生了变化。此外，还测定了ADH-MII的20个N-末端氨基酸，ADH-MI极不稳定，但在约800 mM的硫酸铵存在下保持稳定。该酶是在硫酸铵存在下从乙醇培养的细胞中纯化出来的，是一种电泳均一的蛋白质。该天然酶的相对分子质量约为12万。它由两个亚基(90,000和40,000)组成。乙醇和NAD的Km值分别为1.25×10~(-3)M和6.25×10~(-5)M。低浓度的锌离子对其他利用甲醇的酵母菌、多形汉逊酵母和巴斯德毕赤酵母的乙醇脱氢酶活性也有抑制作用，其Ki值为5.35×10~(-5)。

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+>.