Structure/activity studies of two molybdenum enzymes

描述(申请人提供)：拟开展的工作集中在两种含钼酶的基础结构/活性研究上，即黄嘌呤氧化还原酶和一氧化碳脱氢酶。第一种酶催化人体嘌呤代谢的最后两步，也是治疗高尿酸血症和化疗方案的重要靶点。它已经成为钼羟基酶家族机理研究的范例。第二种酶催化某些细菌中CO的羧基营养生长的关键步骤，负责每年从环境中去除大约2x108公吨的CO。从化学的角度来看，这也是一种重要的生物反应：虽然CO脱氢酶在结构和序列上与黄嘌呤氧化还原酶有显著的同源性，但它在钼羟基酶中的独特之处在于它的反应不涉及C-H键的断裂。拟议工作的总体目标是在其结构的背景下更完整地了解这两种酶的作用机制，其指导假设是酶的功能由活性部位的物理和电子结构决定。具体目标包括快速动力学研究以及旨在确定所确定的中间体的电子结构的光谱工作。在黄嘌呤氧化还原酶的情况下，针对特定活性部位氨基酸残基的定点突变体将被检测，以评估它们在催化中的作用。总体目标是比较和对比这两种密切相关的酶的行为，以便更好地了解结构与功能的关系。

DESCRIPTION (provided by applicant): The proposed work focuses on basic structure/activity studies of two molybdenum-containing enzymes, xanthine oxidoreductase and carbon monoxide dehydrogenase. The first enzyme catalyzes the final two steps in purine metabolism in humans, and is also an important therapeutic target in the treatment of hyperuricemia and also in chemotherapeutic regimens. It has become the paradigm for mechanistic studies of the molybdenum hydroxylase family of enzymes. The second enzyme catalyzes the key step in carboxydotrophic growth on CO in certain bacteria and is responsible for removal of approximately 2 x 108 metric tons of CO from the environment each year. It is also a biological reaction of fundamental interest from a chemical standpoint: although CO dehydrogenase exhibits significant structural and sequence homologies to xanthine oxidoreductase, it is unique among the molybdenum hydroxylases in that its reaction does not involve C-H bond cleavage. The overall goal of the proposed work is to gain a more complete understanding of the mechanism of action of these two enzymes in the context of their structures, the guiding hypothesis being that enzyme function is dictated by the physical and electronic structure of the active site. The Specific Aims include rapid kinetic studies as well as spectroscopic work aimed at determining the electronic structures of intermediates identified. In the case of xanthine oxidoreductase, site-directed mutants targetting specific active site amino acid residues will be examined to evaluate their roles in catalysis. The overall goal is to compare and contrast the behavior of these two closely related enzymes so that the relationship of structure to function can be better understood.