1993 Fiscal Year Final Research Report Summary
Function and structure of peculiar yeast alcohol dehydrogenases of which activities are reversibly inhibited by Zn^<2+>
| Project/Area Number |
04660101
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用微生物学・発酵学
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| Research Institution | Chiba University |
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Principal Investigator |
FUJII Takaki Chiba Univ.Bioproduction Science professor, 園芸学部, 教授 (50125952)
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| Co-Investigator(Kenkyū-buntansha) |
SHINOYAMA Hirofumi Chiba Univ.Bioproduction Science assistant professor, 園芸学部, 助教授 (40211958)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Alcohol dehydrogenase / methanol-utilizing yeast / zinc ion-inhibition / candida sp. |
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| Research Abstract |
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+>.
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