Structure/activity studies of two molybdenum enzymes

描述（由申请人提供）：拟议的工作重点是两种含氘酶（黄嘌呤氧化还原酶和一氧化碳脱氢酶）的基本结构/活性研究。第一种酶催化人类嘌呤代谢的最后两个步骤，也是治疗高尿酸血症和化疗方案中的重要治疗靶点。它已成为钼羟化酶家族的酶的机制研究的范例。第二种酶催化某些细菌在CO上的一氧化碳营养生长的关键步骤，每年负责从环境中去除约2 x 108公吨的CO。从化学观点来看，它也是一种具有根本意义的生物反应：虽然CO脱氢酶与黄嘌呤氧化还原酶具有显著的结构和序列同源性，但它在钼羟化酶中是独特的，因为它的反应不涉及C-H键裂解。拟议的工作的总体目标是获得这两种酶在其结构的背景下的作用机制的更完整的理解，指导假设是酶的功能是由活性位点的物理和电子结构决定的。具体目标包括快速动力学研究以及旨在确定已鉴定中间体电子结构的光谱工作。在黄嘌呤氧化还原酶的情况下，定点突变体靶向特定的活性位点的氨基酸残基将进行检查，以评估其在催化作用。总体目标是比较和对比这两种密切相关的酶的行为，以便更好地理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.