X-RAY HIGH RESOLUTION EMISSION STUDY OF THE BINDING MODE OF CARBOXYLATES AND WAT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 羧基肽酶A和热裂解酶催化多肽键的水解。两者的活性中心都有一个锌与两个组氨酸结合，一个是羧酸分子，一个是溶剂分子。羧酸盐的结合方式因酶的不同而不同，也随pH的不同而变化(单齿到双齿)。对金属结合的氢氧化物的攻击被认为是其中一个假想机制的第一步。因此，测定溶剂的质子化状态对于了解酶的作用机制是至关重要的。由于N从组氨酸和O从溶剂中的散射相相似，XAS不能准确地分辨由于溶剂分子质子化程度不同而导致的金属-溶剂距离的细微差别。此外，锌具有封闭的d壳层，不存在前缘，这通常为分析金属位置的电子结构提供了更多的特征。不同的是，交叉峰的高分辨率X射线发射测量(XES)对质子化的差异非常敏感，正如以前对碳酸酐酶及其模型化合物的研究所看到的那样。在这项工作中，将收集两个不同的pH值(约6和8.5)下的羧肽酶A和嗜热分解酶的交叉峰和Kbeta2，5个峰。利用分子模拟、DFT XANES模拟和真实空间多重散射理论，可以计算占据态密度，并与实验数据进行比较。联合分析可以确定不同pH值下溶剂的质子化状态和羧酸盐的取向，并为讨论酶的机理假说提供坚实的基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The proteases carboxypeptidase A and thermolysin catalyze the hydrolysis of peptide bonds. Both have a Zn in the active site bound to two histidines, one carboxylate and one solvent molecule. The binding mode of the carboxylate differs between the enzymes and also changes depending on pH (monodentate to bidentate). The attack to a metal bound hydroxide has been suggested as the first step in one of the hypothesized mechanisms. Thus, determination of the protonation state of the solvent is essential to understand the mechanism of the enzymes. XAS, due to the similar scattering phases of N from histidine and O from the solvent, cannot accurately discern the subtle difference in the metal-solvent distance due to different protonation of the solvent molecule. Moreover Zn, with its closed d-shell, does not show a pre-edge, which usually provides more features to analyze the electronic structure of the metal site. At difference, high-resolution x-ray emission measurements (XES) of the crossover peak are very sensitive to differences in protonation, as seen in former studies on carbonic anhydrase and its model compounds. In this work the crossover and Kbeta2,5 peaks of the enzymes carboxypeptidase A and thermolysin will be collected for two different pH values (about 6 and 8.5). Using molecular modeling, DFT XANES simulations and also real space multiple scattering theory, the densities of occupied states can be calculated and compared to the experimental data. The combined analysis would allow determining the protonation state of the solvent and the orientation of the carboxylate for the different values of the pH, and provide a solid base to discuss the hypothesis on the mechanisms of the enzymes.