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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 蛋白酶羧肽酶A和嗜热菌蛋白酶催化肽键的水解。两者都在活性位点中具有与两个组氨酸、一个羧酸盐和一个溶剂分子结合的Zn。羧酸盐的结合模式在酶之间不同，并且还根据pH（单齿到双齿）而变化。对金属结合的氢氧化物的攻击被认为是假设机制之一的第一步。因此，确定溶剂的质子化状态对于理解酶的作用机制是必不可少的。XAS，由于类似的散射相的N从组氨酸和O从溶剂，不能准确地辨别金属-溶剂距离的细微差异，由于不同的质子化的溶剂分子。此外，Zn具有封闭的d-壳层，不显示前边缘，这通常提供更多的特征来分析金属位点的电子结构。在差异，高分辨率的X射线发射测量（XES）的交叉峰是非常敏感的质子化的差异，在碳酸酐酶和它的模型化合物的以前的研究中所看到的。在这项工作中，将收集两种不同pH值（约6和8.5）下羧肽酶A和嗜热菌蛋白酶的交叉峰和K β 2，5峰。利用分子模拟、DFT XANES模拟以及真实的空间多重散射理论，可以计算占据态的密度，并与实验数据进行比较。结合分析将允许确定的质子化状态的溶剂和取向的羧酸盐的不同值的pH值，并提供了一个坚实的基础上讨论的酶的机制的假设。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.