THE REDUCTIVE ACTIVATION OF DIOXYGEN BY GLUCOSE OXIDASE

这项拟议的研究旨在帮助阐明酶激活分子氧的一般策略。氧的还原激活存在于广泛的生物过程中，从细胞色素P450和非金属黄素酶的氧化代谢到博莱霉素等化疗药物的激活。最近，人们的注意力转向了黄素和蝶呤等有机辅因子与氧气反应的机制。这项工作将集中在葡萄糖氧化酶(GO)上，这是一种黄素酶，利用分子氧将葡萄糖转化为葡萄糖内酯。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条件下，速率控制步骤是单电子转移。本课程将研究氧气活化的微观步骤，包括控制电子转移速率的参数。通过动力学研究，电子转移距离将被确定，并将与现有的结晶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(S)，这些残基被认为是向初始超氧化物中间体传递静电稳定的信息。因此，提出的酶作用机制将得到验证。此外，还将研究与GO催化有关的氧-18动力学同位素效应(0-18Kie)。将通过使用与合成黄素重组的载脂蛋白酶的系统研究来探索驱动力依赖性。这一结果将为解释在其他体系中观察到的0-18Kie提供一个参考框架，其中指出了单电子转移机制。还将尝试研究0-18 Kie的温度依赖性，从而解决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.