Biochemical Studies of Oxalate Decarboxylase

能够催化草酸分解的酶在治疗草酸方面具有潜在的应用 治疗与这种化合物在血液中积聚有关的人类病理情况 和/或尿液。这项建议概述了综合实验和计算研究的继续。 为了了解草酸脱羧酶(OxDC)的基本生物化学和调控， 催化草酸转化为二氧化碳和甲酸盐的酶。这个项目的目标是 (I)测试自由基物种参与分子过程的机制建议 草酸盐C-C键的断裂，(Ii)研究氧的作用和更高的氧化态 活性中心锰离子在催化机理中的作用，以及(Iii)评估局部蛋白质的变化 环境起作用，从而调节酶中锰(II)中心的内在反应活性。一个完整的 实验和计算策略，使用生物无机化学、分子光谱学、 将学习酶动力学和蛋白质工程、X射线结晶学和计算化学。 这些努力有两个主要的具体目标。在第一个目标，生物物理结构方法，EPR 光谱和密度泛函理论(DFT)计算将用于验证假设 OxDC催化脱羧基的机理，并建立了准确的锰氧化态 调节反应。第二个目标将集中在OxDC的动力学和生物物理特性上。 突变体，以评估蛋白质环境在调节蛋白质的化学反应活性中的作用 酶中的Mn(II)中心(S)。除了这些努力的一般生化影响之外，我们的 了解酶可以催化困难反应的化学机制，以及分子 看似用于进化具有改变催化性质的蛋白质的过程，这些努力应该 为临床新疗法的开发提供长期翻译研究的平台 草酸相关疾病的治疗和/或预防。

Enzymes that can catalyze the breakdown of oxalic acid have potential therapeutic application in the treatment of human pathological conditions associated with the accumulation of this compound in the blood and/or urine. This proposal outlines the continuation of integrated experimental and computational studies aimed at understanding the fundamental biochemistry and regulation of oxalate decarboxylase (OxDC), an enzyme that catalyzes the conversion of oxalate to carbon dioxide and formate. The goals of this project are (i) to test mechanistic proposals for the involvement of radical species in the molecular processes that result in cleavage of the oxalate C-C bond, (ii) to investigate the role of dioxygen and higher oxidation states of the active site manganese ion in the catalytic mechanism, and (iii) to evaluate changes in local protein environment function so as to modulate the intrinsic reactivity of the Mn(II) center in the enzyme. An integrated experimental and computational strategy, using techniques in bioinorganic chemistry, molecular spectroscopy, enzyme kinetics and protein engineering, X-ray crystallography, and computational chemistry, will be pursued in these efforts, which have two major specific aims. In the first aim, biophysical structural methods, EPR spectroscopy and density functional theory (DFT) calculations will be used to validate the hypothetical mechanism of OxDC-catalyzed decarboxylation, and establish the precise manganese oxidation state that mediates the reaction. The second aim will focus on the kinetic and biophysical characterization of OxDC mutants in an effort to evaluate the effect of protein environment in modulating the chemical reactivity of the Mn(II) center(s) in the enzyme. In addition to the general biochemical implications of these efforts for our knowledge of the chemical mechanisms by which enzymes can catalyze difficult reactions, and the molecular processes that seem to be used in evolving proteins with altered catalytic properties, these efforts should provide a platform for long-term translational research on the development of novel therapies for the clinical treatment and/or prevention of oxalate-related disease.