Novel Approach to Study Transient Enzyme Intermediates

研究瞬时酶中间体的新方法

• 批准号: 7009653
• 负责人: Karen S. Anderson
• 金额: $ 29.1万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7009653
• 关键词: active sites; biological signal transduction; chemical kinetics; chemical reaction; chymotrypsin; electrospray ionization mass spectrometry; enzyme activity; phosphorylation; protein structure function; time resolved data

DESCRIPTION (PROVIDED BY APPLICANT) An important aspect of protein structure-function studies and enzyme-targeted biorational inhibitor design is an understanding of how chemical catalysis is occurring a the enzyme active site. The detection and identification of enzyme reaction intermediates provide key insights into the enzyme catalytic reaction pathway. Rapid chemical quench methods involving off-line detection have proven very useful in identifying enzyme reaction intermediates. However, a limitation of this approach involves the inability to detect and characterize enzyme intermediates that are labile under the chemical quenching conditions. We have developed and demonstrated the utility for the real-time detection and characterization of enzyme intermediates on the millisecond time scale using a novel approach of rapid mixing/time-resolved electrospray ionization (ESi) mass spectrometry (MS). The overall objective of the studies described in this proposal is to further develop and demonstrate the broad applicability of the rapid mix/time-resolved ESIMS approach for detecting and quantitating enzyme intermediates and defining enzyme reaction kinetic pathways. The following specific aims are designed to accomplish this objective: 1. Demonstrate the ability of rapid mixing/time-resolved ESI-MS to quantitatively determine kinetic parameters. 2. Demonstrate the broader utility of the rapid mixing/time-resolved ESI-MS to identify putative chemically labile enzyme intermediates. 3. Demonstrate the ability of rapid mixing/time-resolved ESI-MS to monitor specific phosphorylation and dephosphorylation events in protein signaling. These studies will aid in pioneering a powerful and novel analytical methodology for detecting, characterizing and quantitating enzyme intermediates on a rapid time scale.

描述（由申请人提供）蛋白质结构功能研究和酶靶向生物理性抑制剂设计的一个重要方面是了解化学催化如何在酶活性位点发生。酶反应中间体的检测和鉴定为酶催化反应途径提供了重要的见解。涉及离线检测的快速化学猝灭方法已被证明在识别酶反应中间体方面非常有用。然而，这种方法的局限性在于无法检测和表征在化学猝灭条件下不稳定的酶中间体。我们开发并展示了使用快速混合/时间分辨电喷雾电离 (ESi) 质谱 (MS) 的新方法在毫秒时间尺度上实时检测和表征酶中间体的实用性。本提案中描述的研究的总体目标是进一步开发和证明快速混合/时间分辨 ESIMS 方法在检测和定量酶中间体以及定义酶反应动力学途径方面的广泛适用性。为了实现这一目标，设计了以下具体目标： 1. 展示快速混合/时间分辨ESI-MS定量测定动力学的能力 参数。 2. 展示快速混合/时间分辨 ESI-MS 在识别假定的化学不稳定酶中间体方面的更广泛用途。 3. 展示快速混合/时间分辨 ESI-MS 监测蛋白质信号传导中特定磷酸化和去磷酸化事件的能力。 这些研究将有助于开创一种强大而新颖的分析方法来检测、 快速表征和定量酶中间体。