THE REDUCTIVE ACTIVATION OF DIOXYGEN BY GLUCOSE OXIDASE

提出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和非金属含黄酶的氧化代谢到化疗药物如博来霉素的活化。最近，人们的注意力转向了有机辅助因子，如黄素和叶黄素，与二氧反应的机制。这项工作将侧重于葡萄糖氧化酶（GO），一种利用分子氧将葡萄糖转化为葡萄糖酸内酯的黄酶。氧化石墨烯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，其速率控制步骤是单电子转移。微观步骤涉及到二氧活化将被检查，包括控制电子转移速率的参数。从动力学研究中，电子转移距离将被确定，并将与可用的晶体学数据结合使用，以设计定点突变蛋白。诱变研究将允许去除带正电的氨基酸残基，这些残基被认为是将静电稳定传递给初始超氧化物中间体的。因此，提出的酶的作用机制将被测试。此外，氧-18动力学同位素效应（0-18 KIEs）与氧化石墨烯催化将被研究。我们将通过系统的研究，包括用合成黄素重组载脂蛋白酶的使用，来探讨其驱动力依赖性。该结果将为解释在其他系统中观察到的0-18 key提供参考框架，其中单电子转移机制表明。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.