Mass Spectrometry and Enzyme Processes

这种竞争性更新的长期目标是开发允许分析 用质谱仪分析各种酶的作用过程。本文提出的主要领域包括： 1)磷酸己糖异构酶的动力学和催化机理的确定。 2)酪氨酸蛋白磺基转移酶的动力学、底物确定和机理确定。这 还将包括一种新的程序，用于明确确定天然底物上的硫酸盐化部位。 3)底物和抑制剂与新发现的人内硫酸酶HSulf-2相互作用的研究 这些领域中的每一个都使用电喷雾电离(ESI)质谱学(MS)，使用LTQ 离子陷阱或FTICR仪器测量各种动力学参数，如Km，Vmax，Ki，Kcat和 特异度常数。提出了用电喷雾电喷雾质谱(ESI-MS)计算动力学参数的方法 传统的分光光度技术对发色团的需求。同样重要的是 事实上，以前难以处理或难以测量的动力学信息可以使用 在此概述的基于MS的方法。分析硫酸盐生物分子的问题在 质谱学。拟议中的研究将缓解与硫酸盐损失相关的问题 在正离子模式下进行电喷雾电离和MS/MS。类似地，提出了解决问题的方法。 获得有关磷酸异构酶系统的生物学数据；其中之一很难从动力学上进行分类 而且是机械的。。。

The long term goal of this competing renewal is the development of methods that allow for the analysis of various enzymatic processes using mass spectrometry. The major areas proposed herein include: 1) Kinetics and catalytic mechanism determination for phosphohexose-isomerases. 2) Kinetics, substrate determination, and mechanism determination of tyrosylprotein sulfotransferases. This will also include a new procedure for unambiguous determination of sites of sulfation on natural substrates. 3) Investigation of substrate and inhibitor interactions with a newly discovered human endosulfatase, Hsulf-2 Each of these areas employs electrospray ionization (ESI)mass spectrometry (MS)using either an LTQ ion trap or an FTICR instrument to measure various kinetic parameters such as Km, Vmax, Ki, kcat and specificity constants. It is proposed that kinetic parameters can be calculated using ESI-MS thus obviating the need for chromophores as required by traditional spectrophotometric techniques. Equally important is the fact that kinetic information, either previously intractable or difficult to measure, can be gleaned using the MS based methods outlined herein. The problem with analyzing sulfated biomolecules is well recognized in mass spectrometry. The proposed research will alleviate those problems associated with loss of sulfate upon electrospray ionization and MS/MS in the positive ion mode. Similarly, methods are proposedfor obtaining biological data on phosphoisomerase systems; one of which is highly difficult to classify kinetically and mechanistically. . .