SENSITIVITY OF THE ESSENTIAL ZINC-THIOLATE MOIETY OF YEAST ALCOHOL-DEHYDROGENASE TO HYPOCHLORITE AND PEROXYNITRITE
SENSITIVITY OF THE ESSENTIAL ZINC-THIOLATE MOIETY OF YEAST ALCOHOL-DEHYDROGENASE TO HYPOCHLORITE AND PEROXYNITRITE
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DOI：
10.1021/bi00011a008
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发表时间：
1995-03-21
期刊：
影响因子：
2.9
通讯作者：
MCCORD, JM
中科院分区：
文献类型：
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作者：
CROW, JP;BECKMAN, JS;MCCORD, JM
酵母醇脱氢酶活性位点处的锌硫醇盐中心的破坏导致失活和锌释放。活性，锌的释放和硫醇/硫醇盐氧化的测量被用来评估生物相关的氧化剂对醇脱氢酶的影响。用1 mM过氧化氢以1.3 M（-1）s（-1）的速率灭活乙醇脱氢酶。过氧亚硝酸盐，近扩散限制的一氧化氮和超氧化物的反应产物，灭活乙醇脱氢酶的IC 50 = 0.95 μ M时，催化浓度的乙醇脱氢酶亚基（0.074 μ M）存在。通过SIN-1失活的乙醇脱氢酶的分解缓慢、连续地产生过氧亚硝酸盐，其效果与推注添加一样有效。通过两种不同的竞争测定法测定，23 ℃下过氧亚硝酸根与乙醇脱氢酶反应的速率常数分别为2.6 × 10（5）M（-1）s（-1）和5.2 × 10（5）M（-1）s（-1）。与醇脱氢酶的反应代表了过氧亚硝酸根测定的最快反应之一。低氧使乙醇脱氢酶以4 × 10（3）M（-1）s（-1）的速率失活。牛磺酸和次氯酸盐的反应产物牛磺酸氯胺的灭活速率常数仅略慢于2.7 × 10（3）M（-1）s（-1）。锌的释放和硫醇/硫醇氧化与失活的过氧亚硝酸盐或次氯酸盐。在过氧亚硝酸盐或次氯酸盐的浓度下，产生完全失活，每个亚基释放0.85个锌原子，每个亚基氧化3个硫醇/硫醇盐。酵母醇脱氢酶（Zn（1）Cys（2）His（1））的锌-硫醇盐部分与锌指蛋白（Zn（1）Cys（2）His（2））中发现的锌-硫醇盐部分之间的结构相似性表明广泛分布的普遍存在的锌指部分可能是氧化剂诱导的损伤的主要靶点。
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.