...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >General Base-General Acid Catalysis in Human Histone Deacetylase 8
【24h】

General Base-General Acid Catalysis in Human Histone Deacetylase 8

机译：一般碱-一般酸催化在人组蛋白脱乙酰酶8中

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

Histone deacetylases (HDACs) regulate cellular processes such as differentiation and apoptosis and are targeted by anticancer therapeutics in development and in the Clinic. HDAC8 is a metal-dependent class I HDAC and is proposed to use a general acid base catalytic pair in the mechanism of amide bond hydrolysis. Here, we report site-directed mutagenesis and enzymological measurements to elucidate the catalytic mechanism of HDAC8. Specifically, we focus on the catalytic function of Y306 and the histidine-aspartate dyads H142-D176 and H143-D183. Additionally, we report X-ray crystal structures of four representative HDAC8 mutants: D176N, D176N/Y306F, D176A/Y306F, and H142A/Y306F. These structures provide a useful framework for understanding enzymological measurements. The pH dependence of k(cat)/K-M for wild-type Co(II)-HDAC8 is bell-shaped with two pK(a) values of 7.4 and 10.0. The upper pK(a) reflects the ionization of the metal-bound water molecule and shifts to 9.1 in Zn(II)-HDAC8. The H142A mutant has activity 230-fold lower than that of wild-type HDAC8, but the pK(al) value is not altered. Y306F HDAC8 is 150-fold less active than the wild-type enzyme; crystal structures show that Y306 hydrogen bonds with the zinc-bound substrate carbonyl, poised for transition state stabilization. The H143A and H142A/H143A mutants exhibit activity that is >80000-fold lower than that of wild type HDAC8; the buried D176N and D176A mutants have significant catalytic effects, with more subtle effects caused by D183N and D183A. These enzymological and structural studies strongly suggest that H143 functions as a single general base general acid catalyst, while H142 remains positively charged and serves as an electrostatic catalyst for transition state stabilization.
机译：组蛋白脱乙酰酶 （HDAC） 调节细胞过程，如分化和凋亡，是开发中和临床上抗癌疗法的靶点。HDAC8 是一种金属依赖性 I 类 HDAC，建议在酰胺键水解机理中使用通用酸碱催化对。在这里，我们报告了定点诱变和酶学测量，以阐明 HDAC8 的催化机制。具体来说，我们专注于 Y306 和组氨酸-天冬氨酸二元组 H142-D176 和 H143-D183 的催化功能。此外，我们报道了四种代表性 HDAC8 突变体的 X 射线晶体结构：D176N、D176N/Y306F、D176A/Y306F 和 H142A/Y306F。这些结构为理解酶学测量提供了一个有用的框架。k（cat）/K-M 对野生型 Co（II）-HDAC8 的 pH 依赖性呈钟形，两个 pK（a） 值分别为 7.4 和 10.0。上部 pK（a） 反映了金属结合水分子的电离，并在 Zn（II）-HDAC8 中移至 9.1。H142A 突变体的活性比野生型 HDAC8 低 230 倍，但 pK（al） 值没有改变。Y306F HDAC8 的活性比野生型酶低 150 倍;晶体结构表明，Y306与锌结合的底物羰基氢键，有望实现过渡态稳定。H143A 和 H142A/H143A 突变体的活性比野生型 HDAC8 低 >80000 倍;埋藏的D176N和D176A突变体具有显著的催化作用，D183N和D183A引起的催化作用更为微妙。这些酶学和结构学研究强烈表明，H143 作为单一的一般碱一般酸催化剂发挥作用，而 H142 保持带正电并作为过渡态稳定的静电催化剂。

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