...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >Kinetic and Spectroscopic Characterization of Type II Isopentenyl Diphosphate Isomerase from Thermus thermophilus: Evidence for Formation of Substrate-Induced Flavin Species
【24h】

Kinetic and Spectroscopic Characterization of Type II Isopentenyl Diphosphate Isomerase from Thermus thermophilus: Evidence for Formation of Substrate-Induced Flavin Species

机译：嗜热栖热菌II型异戊烯基二磷酸异构酶的动力学和光谱表征：底物诱导黄素物种形成的证据。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Type II isopentenyl diphosphate (IPP) isomerase catalyzes the interconversion of IPP and dimethylallyl diphosphate (DMAPP). Although the reactions catalyzed by the type II enzyme and the well-studied type I IPP isomerase are identical, the type II protein requires reduced flavin for activity. The chemical mechanism, including the role of flavin, has not been established for type II IPP isomerase. Recombinant type II IPP isomerase from Thermus thermophilus HB27 was purified by Ni~(2+) affinity chromatography. The aerobically purified enzyme was inactive until the flavin cofactor was reduced by NADPH or dithionite or photochemically. The inactive oxidized flavin-enzyme complex bound IPP in a Mg~(2+)-dependent manner for which K_D ~ K_m~(IIP), suggesting that the substrate binds to the inactive oxidized and active reduced forms of the protein with similar affinities. N,N-Dimethyl-2-amino-1-ethyl diphosphate (NIPP), a transition state analogue for the type I isomerase, competitively inhibits the type II enzyme, but with a much lower affinity. pH-dependent spectral changes indicate that the binding of IPP, DMAPP, and a saturated analogue isopentyl diphosphate promotes protonation of anionic reduced flavin. Electron paramagnetic resonance (EPR) and UV-visible spectroscopy show a substrate-dependent accumulation of the neutral flavin semiquinone during both the flavoenzyme reduction and reoxidation processes in the presence of IPP and related analogues. Redox potentials of IPP-bound enzyme indicate that the neutral semiquinone state of the flavin is stabilized thermodynamically relative to free FMN in solution.
机译：II型异戊烯基二磷酸（IPP）异构酶催化IPP和二甲基烯丙基二磷酸（DMAPP）的相互转化。尽管由II型酶和经过充分研究的I型IPP异构酶催化的反应是相同的，但II型蛋白的黄素活性降低。 II型IPP异构酶的化学机理（包括黄素的作用）尚未确定。用Ni〜（2+）亲和色谱法纯化嗜热栖热菌HB27的II型IPP异构酶。需氧纯化的酶是无活性的，直到黄素辅因子被NADPH或连二亚硫酸盐或光化学还原。无活性的氧化黄素酶复合物以Mg〜（2+）依赖的方式结合IPP，K_D〜K_m〜（IIP）表示底物以相似的亲和力与蛋白的无活性氧化和活性还原形式结合。 N，N-二甲基-2-氨基-1-乙基二磷酸酯（NIPP）是I型异构酶的过渡态类似物，竞争性抑制II型酶，但亲和力低得多。 pH依赖性光谱变化表明IPP，DMAPP和饱和类似物异戊基二磷酸酯的结合可促进阴离子还原黄素的质子化。电子顺磁共振（EPR）和紫外线可见光谱显示，在存在IPP和相关类似物的情况下，黄素半醌在黄素酶还原和再氧化过程中均依赖于底物，积累。 IPP结合酶的氧化还原电势表明黄素的中性半醌状态相对于溶液中的游离FMN热力学稳定。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

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