Inactivation of glyceraldehyde-3-phosphate dehydrogenase and yeast alcohol dehydrogenase by arene oxides.
Inactivation of glyceraldehyde-3-phosphate dehydrogenase and yeast alcohol dehydrogenase by arene oxides.
复制标题
芳烃氧化物使 3-磷酸甘油醛脱氢酶和酵母醇脱氢酶失活。
DOI：
10.1021/bi00602a013
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发表时间：
1978
期刊：
影响因子：
2.9
通讯作者：
T. C. Bruice
中科院分区：
文献类型：
--
作者：
P. Y. Bruice;S. C. Wilson;T. C. Bruice
Paula Yurkanis Bruice、Susan Grase Wilson和托马斯C. Bruice* 摘要：酵母醇脱氢酶（YADH）和兔肌甘油醛-3-磷酸脱氢酶（GPDH）是催化活性所必需的含巯基的酶，它们被几种低浓度的芳烃氧化物（Mis ~ 10~ 4 M）迅速灭活。芳烃氧化物的缓蚀效果依次为：对叔丁氧基羰基苯氧化物>对羧基苯氧化物>菲9，10-氧化物。在pH7.0 -9.5范围内，随着芳烃氧化物浓度的增加和培养液pH值的增加，酶活性的半衰期减小。预先将酶与饱和浓度的底物孵育降低了芳烃氧化物的失活速率，并且将GPDH与芳烃氧化物孵育导致在360 nm处的Racker带的吸收增加约5 h，随后吸收减少。这些观察结果表明，烷基化的活性位点巯基已经发生。用5，5 '-二硫代双（2-硝基苯甲酸）滴定表明，芳烃氧化物对活性中心半胱氨酸残基具有专一性。4-叔丁氧羰基苯氧化物作为YADH和GPDH活性抑制剂的更大有效性是从其相对大的亲核敏感性指数预测的。从Br^ nsted图中得到的硫醇根阴离子与芳烃氧化物反应的Br^ nsted值表明，4-叔丁氧基羰基苯氧化物的亲核进攻位点不同于其它研究的芳烃氧化物。尽管溶菌酶被某些结构上与底物相关的二苯醚和环氧化物抑制，但菲9，10-氧化物对酶的活性没有影响。膜结合细胞色素P-450单加氧酶将芳烃转化为芳烃氧化物（Tomaszewski等人，1974），其在芳香族化合物解毒（Oesch，1973; Jeffrey和Jerina，1975）以及在代谢上重要的酚类的生物合成（Daly等人，1972年）。某些芳烃氧化物已被证明是致癌作用的病原体（Grover等人，1971; Levin等人，1976）、诱变（艾姆斯等人. 1972年）。和坏死（Brodie等人，1971），这是由于它们与细胞大分子共价结合。目前，尚不清楚连接的关键位点是蛋白质还是核酸。几个研究者（Grover和西姆斯，1970和1972; Jeffrey等人，1976 a，B; Blobstein等人，1975，1976）已经确定K-区芳烃氧化物与具有嘌呤碱基（特别是鸟嘌呤）的DNA和RNA共价结合，表现出对芳烃氧化物的最大反应性。然而，迄今为止，基本上没有关于芳烃氧化物与蛋白质相互作用的数据。已经表明（Bruice等人，1976 a，B），在水溶液中芳烃氧化物重排为酚（路径A），并被亲核试剂攻击，主要得到反式加成产物（路径B）。硫醇根阴离子表现出相当大的0+ h+，z-> ZA< C路径A
Paula Yurkanis Bruice, Susan Grase Wilson, and Thomas C. Bruice* abstract: Yeast alcohol dehydrogenase (YADH) and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GPDH), enzymes that contain sulfhydryl groups essential to their cat-alytic activity, have been found to be rapidly inactivated by small concentrations of several arene oxides with Mis of~ 10~ 4 M. Theeffectiveness of the arene oxides as inhibitors decreases in the order: 4-carbo-tm-butoxybenzene oxide> 4-carbox-ybenzene oxide> phenanthrene 9, 10-oxide. The half-life of enzymatic activity decreases with increasing arene oxide concentration and increasing pH of the incubationmixture in the pH range 7.0-9.5. Prior incubation of the enzymes with a saturating concentration of substrate decreases the rate of inactivation by the arene oxides, andincubation of GPDH with arene oxide results in increased absorption of the Racker band at 360 nm for~ 5 h followed by a decrease inabsorption. These observations suggest that alkylation of the active-site sulfhydryl groups has occurred. Titration with 5, 5'-dithiobis (2-nitro-benzoic acid) shows that the arene oxides show specificity toward the active-site cysteineresidues. The greater effectiveness of 4-carbo-iert-butoxybenzene oxide as an inhibitor of YADH and GPDH activity is as would be predicted from its comparatively large nucleophilic susceptibility index. The ß values obtained from Br^ nsted plots for the reaction of thiolate anions with the arene oxides suggest that the site of nucleophilic attack on 4-carbo-tcrt-butoxybenzene oxide is different from that of other investigated arene oxides. Although lysozyme is inhibited by certain diphenyl ethers and epoxides structurally related to the substrate, phenanthrene 9, 10-oxide has no effect on the activity of the enzyme. embrane-bound cytochrome P-450 monoxygenases convert aromatic hydrocarbons to arene oxides (Tomaszewski et al., 1974) which serve as intermediates in aromatic com-pound detoxification (Oesch, 1973; Jeffrey and Jerina, 1975) as well as in the biosynthesis of metabolically important phenols (Daly et al., 1972). Certain arene oxides have been shown to be causative agents of carcinogenesis (Grover et al., 1971; Levin et al., 1976), mutagenesis (Ames et al.. 1972). and ne-crosis (Brodie et al., 1971) as a result of their being bound covalently to cellular macromolecules. At present, it is not known whether the critical site of attachment is protein or nucleic acid. Several investigators (Grover and Sims, 1970 and 1972; Jeffrey et al., 1976a, b; Blobstein et al., 1975, 1976) have established that K-region arene oxides bind covalently to DN A and RNA with the purine bases, in particular guanine, ex-hibiting the greatest reactivity toward the arene oxides. To date, however, there is essentially no data available on the in-teraction of arene oxides with proteins. It has been shown (Bruice et al., 1976a, b) that in aqueous solution arene oxides rearrange to phenols (path A) and are attacked by nucleophiles to give primarily trans-addition products (path B). Thiolate anions exhibit considerably greater 0+ h+, z-> ZA< C path A