...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >Detection and Characterization of Two ATP-Dependent Conformational Changes in Proteolytically Inactive Escherichia coli Lon Mutants by Stopped Flow Kinetic Techniques
【24h】

Detection and Characterization of Two ATP-Dependent Conformational Changes in Proteolytically Inactive Escherichia coli Lon Mutants by Stopped Flow Kinetic Techniques

机译：通过停止流动动力学技术检测和表征蛋白水解惰性大肠杆菌突变体中两个ATP依赖性构象变化。

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

Lon is an ATP dependent serine protease responsible for degrading denatured, oxidatively damaged and certain regulatory proteins in the cell. In this study we exploited the fluorescence properties of a dansylated peptide substrate (S4) and the intrinsic Trp residues in Lon to monitor peptide interacting with the enzyme. We generated two proteolytically inactive Lon mutants, S679A and S679W, where the active site serine is mutated to an Ala and Trp residue, respectively. Stopped-flow fluorescence spectroscopy was used to identify key enzyme intermediates generated along the reaction pathway prior to peptide hydrolysis. A two-step peptide binding event is detected in both mutants, where a conformational change occurs after a rapid equilibrium peptide binding step. The K_d for the initial peptide binding step determined by kinetic and equilibrium binding techniques is approximately 164 micromolar and 38 micromolar, respectively. The rate constants for the conformational change detected in the S679A and S679W Lon mutants are 0.74 亇 0.10 s~(-1) and 0.57 亇 0.10 s~(-1), respectively. These values are comparable to the lag rate constant determined for peptide hydrolysis (k_(lag) approx 1 s~(-1)) [Vineyard, D., et al. (2005) Biochemistry 45, 4602-4610]. Replacement of the active site Ser with Trp (S679W) allows for the detection of an ATP-dependent conformational change within the proteolytic site. The rate constant for this conformational change is 7.6 亇1.0 s~(-1), and is essentially identical to the burst rate constant determined for ATP hydrolysis under comparable reaction conditions. Collectively, these kinetic data support a mechanism by which the binding of ATP to an allosteric site on Lon activates the proteolytic site. In this model, the energy derived from the binding of ATP minimally supports peptide cleavage by allowing peptide substrate access to the proteolytic site. However, the kinetics of peptide cleavage are enhanced by the hydrolysis of ATP.
机译：Lon是一种ATP依赖性丝氨酸蛋白酶，负责降解细胞中变性，氧化损伤的某些调节蛋白。在这项研究中，我们利用了丹磺酰化肽底物（S4）的荧光特性和Lon中固有的Trp残基来监测肽与酶的相互作用。我们生成了两个蛋白水解失活的Lon突变体S679A和S679W，其中活性位点丝氨酸分别突变为Ala和Trp残基。终止流荧光光谱法用于鉴定肽水解之前沿反应路径生成的关键酶中间体。在两个突变体中均检测到两步肽结合事件，其中在快速平衡肽结合步骤后发生构象变化。通过动力学和平衡结合技术确定的初始肽结合步骤的K_d分别约为164微摩尔和38微摩尔。在S679A和S679W Lon突变体中检测到的构象变化速率常数分别为0.74亇0.10 s〜（-1）和0.57亇0.10 s〜（-1）。这些值可与肽水解测定的滞后速率常数（k_（滞后）约1 s〜（-1））相当[Vineyard，D.等。 （2005）Biochemistry 45，4602-4610]。用Trp（S679W）替换活性位点Ser可以检测蛋白水解位点内ATP依赖的构象变化。此构象变化的速率常数为7.6亇1.0 s〜（-1），与在可比较的反应条件下为ATP水解测定的破裂速率常数基本相同。这些动力学数据共同支持一种机制，通过该机制，ATP与Lon的变构位点结合会激活蛋白水解位点。在该模型中，通过允许肽底物进入蛋白水解位点，源自ATP结合的能量极少支持肽裂解。然而，通过ATP的水解增强了肽裂解的动力学。

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