SENSITIVITY OF THE ESSENTIAL ZINC-THIOLATE MOIETY OF YEAST ALCOHOL-DEHYDROGENASE TO HYPOCHLORITE AND PEROXYNITRITE

被引:230
作者
CROW, JP
BECKMAN, JS
MCCORD, JM
机构
[1] UNIV ALABAMA,DEPT BIOCHEM,BIRMINGHAM,AL 35233
[2] UNIV COLORADO,WEBB WARING INST BIOMED RES,DENVER,CO 80262
关键词
D O I
10.1021/bi00011a008
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Disruption of the zinc-thiolate center at the active site of yeast alcohol dehydrogenase results in inactivation and zinc release. Measurements of activity, zinc release, and thiol/thiolate oxidation were used to assess the effects of biologically relevant oxidants on alcohol dehydrogenase. Alcohol dehydrogenase was inactivated by 1 mM hydrogen peroxide at a rate of 1.3 M(-1) s(-1). Peroxynitrite, the near diffusion-limited reaction product of nitric oxide and superoxide, inactivated alcohol dehydrogenase with an IC50 = 0.95 mu M when catalytic concentrations of alcohol dehydrogenase subunit (0.074 mu M) were present. Slow, continuous production of peroxynitrite from decomposition of SIN-1 inactivated alcohol dehydrogenase as effectively as bolus addition. The rate constants for reaction of peroxynitrite with alcohol dehydrogenase at 23 degrees C as determined by two different competition assays were 2.6 x 10(5) M(-1) s(-1) and 5.2 x 10(5) M(-1) s(-1). The reaction with alcohol dehydrogenase represents one of the fastest reactions yet determined for peroxynitrite. Hypochlorite inactivated alcohol dehydrogenase at a rate of 4 x 10(3) M(-1) s(-1). The rate constant for inactivation by taurine choramine, the reaction product of taurine and hypochlorite, was only slightly slower at 2.7 x 10(3) M(-1) s(-1). Zinc release and thiol/thiolate oxidation were correlated with inactivation by either peroxynitrite or hypochlorite. At the concentrations of peroxynitrite or hypochlorite producing total inactivation, 0.85 zinc atom was released per subunit and 3 thiol/thiolates per subunit were oxidized. The structural similarity between the zinc-thiolate moiety of yeast alcohol dehydrogenase (Zn(1)Cys(2)His(1)) and that found in zinc finger proteins (Zn(1)Cys(2)His(2)) suggest that the widely distributed ubiquitous zinc finger moiety may be a major target for oxidant-induced injury.
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页码:3544 / 3552
页数:9
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