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

Zn^<2>可逆抑制活性的特有酵母醇脱氢酶的功能和结构

基本信息

批准号：
04660101
负责人：
FUJII Takaki
金额：
$ 1.28万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

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+>.

我们发现，利用甲醇的酵母，假丝酵母属N-16，有两种类型的NAD依赖性乙醇脱氢酶。一种是组成型酶（ADH-MI），另一种是诱导型酶（ADH-MII）。Zn^<2+>对这些酶有抑制作用，用圆二色性（CD）分析法检测了Zn^<2+>对ADH-MII纯化产物的影响。其CD谱与马肝和面包酵母中的ADH不同。这些光谱数据表明，在Zn^<2+>存在下，ADH-MII的构象发生了变化。ADH-MII的N-端氨基酸序列分析表明，ADH-MII极不稳定，但在硫酸铵（约800 mM）存在下仍保持稳定。该酶在硫酸铵存在下纯化为来自在乙醇上生长的细胞的电泳均一蛋白。天然酶的分子量估计为约120，000。它由两个分股（90 000和40 000）组成。乙醇和NAD的Km值分别为1.25 × 10 - <-3>4 M和6.25 × 10 - 4 <-5>M。Zn^<2+>的Ki值为5.35 × 10^<-5>M。从其他利用甲醇的酵母（多形汉逊酵母和巴斯德毕赤酵母）制备的无细胞提取物中的醇脱氢酶活性也被低浓度的Zn^<2+>抑制。