Mechanistic Studies on F420 dependent Glucose-6-Phosphate Dehydrogenase from Mycobacteria tuberculosis

摘要：F420依赖性葡萄糖-6-磷酸脱氢酶（FGD）是一种在体内发现的必需酶 结核分枝杆菌，结核病的病原体。由FGD催化的反应是 葡萄糖-6-磷酸（G6 P）转化为6-磷酸葡糖内酯。底物G6 P提供电子 减少F420辅因子。本研究的最初目标是表征氢化物转移 采用稳态和准稳态动力学方法对烟气脱硫反应机理进行了研究。我们初步的动能分析 wtFGD的研究，包括一系列的实验，以解决这一目标。具体目标1建议使用 定点诱变，沿着结合研究，稳态和预稳态动力学实验， 一种研究保守活性位点氨基酸残基功能的方法。我们最近解决了 FGD与其底物G6 P复合的晶体结构。这种晶体结构沿着与以前的 已发表的结构暗示了几个保守的活性位点残基，它们可能参与氢化物中 FGD转移反应。然而，通过动力学询问这些氨基酸的能力受到限制 到现在本研究的目的是为阐明烟气脱硫机理提供实验数据。PI的 一个实验室已经创造了几个这样的FGD变体，其中之一是H40 A。一个共同的目标是 进行循环伏安实验，以观察活性位点突变对电位的影响 关于F420 Cofactor具体目标2是研究FGD中的氢化物隧穿。氢化物的研究 近年来，在酶中的隧穿变得更加普遍，并已被认为这是一种 酶的催化策略，驱动氢转移反应。一般来说，对这一问题的调查 F420辅因子依赖性酶的类型尚未被研究。具体目标2的目标是使用同位素 标记，预稳态和稳态动力学分析，以研究FGD中的氢化物隧穿。具体目标 3是利用溶剂同位素效应实验进行质子清查，确定 FGD内的可交换质子是活性的。总而言之，该提案描述了基础研究 为了阐明一种重要的天然结核酶的动力学和机制， 目前可获得的此类数据相对较少。

Abstract: F420-dependent glucose-6-phosphate dehydrogenase (FGD), is an essential enzyme found in Mycobacterium tuberculosis, the causative agent of tuberculosis disease. The reaction catalyzed by FGD is the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone. The substrate, G6P donates electrons for the reduction of the F420 Cofactor. The initial goal of this study is to characterize the hydride transfer reaction mechanism of FGD using steady state and pre-steady state kinetic methods. Our preliminary kinetic studies with wtFGD, includes a series of experiments that address this goal. Specific aim 1 proposes to use site-directed mutagenesis, along with binding studies, steady-state and pre-steady state kinetic experiments as a means to investigate the functionality of conserved active site amino acid residues. We have recently solved the crystal structure of FGD in complex with its substrate, G6P. This crystal structure along with a previously published structure implicates several conserved active site residues that may participate within the hydride transfer reaction of FGD. However, the ability to interrogate these amino acids via kinetics has been limited until now. The goal of this aim is to provide experimental data to elucidate the mechanism of FGD. The PI's laboratory has already created several of these FGD variants, one of which is H40A. A concurrent goal is to conduct cyclic voltammetry experiments in order to observe the effects of active site mutations on the potential of the F420 Cofactor. Specific aim 2 is to investigate hydride tunneling within FGD. The study of hydride tunneling, in enzymes has become more prevalent in recent years and has been suggested that this is a catalytic strategy for enzymes that drive hydrogen transfer reactions. Generally speaking, investigations of this type have not been pursued for F420 Cofactor dependent enzymes. The goal of specific aim 2 is to use isotopic labeling, pre-steady state and steady-state kinetic analysis to study hydride tunneling within FGD. Specific aim 3 is to utilize solvent isotope effects experiments to conduct a proton inventory to determine the number of exchangeable protons within FGD that are active. In summation, this proposal describes fundamental studies to elucidate the kinetics and mechanism of an important naturally occurring tuberculosis enzyme, but one for which relatively little such data are currently available.