• 主题
高级搜索  >
历史搜索 清空历史
首页> 美国卫生研究院文献>Biochemical Journal >Overexpression of restructured pyruvate dehydrogenase complexes and site-directed mutagenesis of a potential active-site histidine residue.
【2h】

Overexpression of restructured pyruvate dehydrogenase complexes and site-directed mutagenesis of a potential active-site histidine residue.

机译：重组丙酮酸脱氢酶复合物的过表达和潜在活性位点组氨酸残基的定点诱变。

代理获取
本网站仅为用户提供外文OA文献查询和代理获取服务，本网站没有原文。下单后我们将采用程序或人工为您竭诚获取高质量的原文，但由于OA文献来源多样且变更频繁，仍可能出现获取不到、文献不完整或与标题不符等情况，如果获取不到我们将提供退款服务。请知悉。
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase multienzyme complex (PDH complex). A thermoinducible expression system was developed to amplify a variety of genetically restructured PDH complexes, including those containing three, two, one and no lipoyl domains per E2p chain. Although large quantities of the corresponding complexes were produced, they had only 20-50% of the predicted specific activities. The activities of the E1p components were diminished to the same extent, and this could account for the shortfall in overall complex activity. Thermoinduction was used to express a mutant PDH complex in which the putative active-site histidine residue of the E2p component (His-602) was replaced by cysteine in the H602C E2p component. This substitution abolished dihydrolipoamide acetyltransferase activity of the complex without affecting other E2p functions. The results support the view that His-602 is an active-site residue. The inactivation could mean that the histidine residue performs an essential role in the acetyltransferase reaction mechanism, or that the reaction is blocked by an irreversible modification of the cysteine substituent. Complementation was observed between the H602C PDH complex and a complex that is totally deficient in lipoyl domains, both in vitro, by the restoration of overall complex activity in mixed extracts, and in vivo, from the nutritional independence of strains that co-express the two complexes from different plasmids.
机译：大肠杆菌的aceEF-lpd操纵子编码丙酮酸脱氢酶多酶复合物（PDH复合物）的丙酮酸脱氢酶（E1p），二氢脂酰胺乙酰转移酶（E2p）和二氢脂酰胺脱氢酶（E3）成分。开发了一种热诱导表达系统，以扩增多种基因重组的PDH复合物，包括每个E2p链包含三个，两个，一个和无脂酰结构域的PDH复合物。尽管产生了大量相应的复合物，但它们仅具有预测的比活的20-50％。 E1p成分的活性降低了相同程度，这可以解释整个复杂活动的不足。热诱导用于表达突变的PDH复合物，其中E2p组分（His-602）的假定的活性位点组氨酸残基被H602C E2p组分中的半胱氨酸代替。该取代消除了该复合物的二氢脂酰胺乙酰转移酶活性，而没有影响其他E2p功能。结果支持了His-602是活性位点残基的观点。失活可能意味着组氨酸残基在乙酰转移酶反应机理中起重要作用，或者该反应被半胱氨酸取代基的不可逆修饰所阻断。观察到H602C PDH复合物与完全缺乏脂酰结构域的复合物之间的互补作用，无论是在体外，还是通过混合表达两种菌株共同表达的菌株的营养独立性，在混合提取物中恢复了整体复合物的活性。不同质粒的复合物。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
代理获取

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号