THE REDUCTIVE ACTIVATION OF DIOXYGEN BY GLUCOSE OXIDASE

拟议的研究旨在帮助阐明酶激活分子氧的一般策略。双氧的还原活化发生在广泛的生物过程中，从细胞色素P450和非金属含黄素酶的氧化代谢到化学治疗剂如博来霉素的活化。最近，注意力转向有机辅因子（如黄素和蝶呤）与分子氧反应的机制。这项工作将集中在葡萄糖氧化酶（GO），一种黄素酶，使用分子氧将葡萄糖转化为β-内酯。GO催化的氧化相特别适合研究，因为它在动力学上不复杂，特别是在高pH下，其中速率控制步骤是单电子转移。将检查分子氧活化所涉及的微观步骤，包括控制电子转移速率的参数。从动力学研究中，将确定电子转移距离，并将其与可用的晶体学数据一起用于设计定点突变蛋白质。诱变研究将允许去除带正电荷的氨基酸残基，这些氨基酸残基被认为与初始超氧化物中间体具有静电稳定性。因此，将测试所提出的酶作用机制。此外，将研究与GO催化相关的氧-18动力学同位素效应（0-18 KIE）。驱动力的依赖性将通过一个系统的研究，涉及使用合成黄素重组的载脂蛋白酶进行探讨。结果将提供一个参考框架的解释0-18 KIE在其他系统中观察到的单电子转移机制表示。还将尝试研究0- 18 KIE的温度依赖性，从而解决在还原过程中核隧穿的可能性。

The proposed research is designed to help illuminate the general strategies used by enzymes to activate molecular oxygen. Reductive activation of dioxygen occurs in a wide range of biological processes, from oxidative metabolism by cytochrome P450s and non-metal- containing flavoenzymes to activation of chemotherapeutic agents like bleomycin. Recently attention has turned to the mechanisms by which organic cofactors, like flavins and pterins, react with dioxygen. This work will focus on glucose oxidase (GO), a flavoenzyme that uses molecular oxygen to convert glucose to gluconolactone. The oxidative phase of GO catalysis is particularly amenable to study because it is kinetically uncomplicated, especially at high pH where the rate- controlling step is a single electron transfer. The microscopic steps involved in dioxygen activation will be examined including the parameters that control electron transfer rates. From kinetic studies the electron transfer distance will be determined and will be used, in conjunction with the available crystallographic data, to design site- directed mutant proteins. The mutagenesis studies will allow removal of positively charged amino acid residue(s) which are believed to communicate electrostatic stabilization to the incipient superoxide intermediate. Thus, the proposed mechanism of enzyme action will be tested. In addition, the oxygen-18 kinetic isotope effects (0-18 KIEs) associated with GO catalysis will be investigated. The driving-force dependence will be probed through a systematic study involving the use of apo enzyme reconstituted with synthetic flavins. The results will provide a frame of reference for the interpretation of 0-18 KIEs observed in other systems where single-electron transfer mechanisms are indicated. Attempts will also be made to study the temperature dependence of the 0- 18 KIE and, thereby, address the possibility of nuclear tunneling during the reduction process.