GEOMETRIC INFLUENCES ON THE PROTONATION STATE OF ZINC-COORDINATED INHIBITORS IN

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 基质金属蛋白酶（MMP）包括锌依赖性蛋白水解酶家族，其降解和重塑细胞外基质（细胞之间的空间）的所有主要组分。发挥如此广泛的生理作用，MMPs已涉及许多疾病，包括癌症，关节炎和心血管疾病。然而，在过去的二十年里，人们在控制它们方面付出了很多努力，却没有得到多少回报。所有MMP中的催化位点由与3个组氨酸氨基酸（残基）的侧链氮原子配位的锌离子组成。第四个配位位点被产生[（His）3 Zn（II）-L]（L= OH 2）催化中心的酶的活性形式的水分子占据。这个水分子取代了半胱氨酸硫原子，半胱氨酸硫原子结合在失活的酶中，或者被抑制剂的锌结合基团取代，抑制剂是一种停止催化的分子。已知的关键质子转移步骤发生在MMP的催化作用机制和活化步骤中，并且也被认为发生在抑制过程中。该提议涉及MMP活性位点的理论计算，假设锌离子的结构变化对这些质子转移过程的能量有显著影响，如所概述的：为了检查MMP活性位点的质子化状态的几何影响，将对大MMP活性位点模型进行混合量子力学/半经验计算。该模型将涵盖催化结构域保守残基的整个HExGHxxGxxH序列。HExGHxxGxxH序列是所有MMP的标志性锌结合基序，催化锌离子与这3个组氨酸（H）残基配位。锌离子、三个配位的组氨酸、谷氨酸（序列中的E）和第四个锌配位的配体（L = OH 2或SHCH 3）将使用密度泛函理论或MP2理论处理，而序列的其余部分使用半经验PM 3方法处理。锌的几何形状的锌结合配体的质子化状态的影响将进行分析，并确定关键的相互作用，详细说明了锌的几何形状与质子化状态的关系的分子基础。混合计算将利用高斯03软件中采用的ONIOM技术，计算将由我和1或2名本科研究人员进行。

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. Matrix metalloproteinases (MMPs) comprise a family of zinc-dependent proteolytic enzymes that degrade and remodel all the major components of the extracellular matrix, the space between the cells. Playing such a broad physiological role the MMPs have been implicated in a host of diseases including cancer, arthritis, and cardiovascular disease. However, over the last two decades much effort and little reward have been realized toward their control. The catalytic site in all MMPs consists of a zinc ion coordinated to side chain nitrogen atoms of 3 histidine amino acids(residues). The fourth coordination site is occupied by a water molecule in the active form of the enzyme producing the [(His)3Zn(II)-L] (L=OH2) catalytic center. This water molecule replaces a cysteine sulfur atom, which is bound in the inactive enzyme, or is replaced by the zinc binding group of an inhibitor, a molecule that stops catalysis. Known critical proton transfer steps occur in the MMP mechanism of catalytic action and activation steps, and are also thought to occur during inhibition. This proposal involves theoretical calculations of the MMP active site, with the hypothesis that structural changes about the zinc ion have a significant impact on the energetics of these proton transfer processes, as outlined: To examine the geometric influences on protonation state of the MMPs active site, hybrid quantum mechanical/semi-empirical calculations will be performed on large MMP active site models. The model will encompass the entire HExGHxxGxxH sequence of conserved residues of the catalytic domain. The HExGHxxGxxH sequence is the signature zinc-binding motif of all MMPs, with the catalytic zinc ion coordinated to these 3 histidine (H) residues. The zinc ion, the three coordinated histidines, the glutamate (E in the sequence), and the fourth zinc coordinated ligand (L = OH2 or SHCH3), will be treated using density functional theory or MP2 theory, while the rest of the sequence is treated using the semi-empirical PM3 method. Zinc geometry influences on the protonation state of the zinc bound ligands will be analyzed, and key interactions will be identified detailing the molecular basis for the zinc geometry versus protonation state relationship. The hybrid calculations will utilize the ONIOM technique employed in the Gaussian 03 software and the computations will be carried out by myself and 1 or 2 undergraduate researchers.