Biochemistry of oxygenases: Mechanistic studies of a cofactor-independent, CO-formingm dioxygenase belonging to the a/ß-hydrolase fold family

2-甲基-3-羟基-4（1H）喹诺酮2，4-双加氧酶（2-Methyl-3-hydroxy-4（1H）quinolone 2，4-dioxygenase，Hod）是一种非辅酶依赖性双加氧酶，具有α/β-水解酶折叠，催化其有机底物裂解为一氧化碳和N-乙酰邻氨基苯甲酸。该项目的总体目标是促进对无辅因子加氧酶如何工作的全面理解，并从机械上比较不依赖辅因子的加氧酶与依赖金属和黄素的加氧酶。将进行瞬态动力学研究，以深入了解Hod催化反应的时间依赖性微观途径。结合现有的结构数据（R。Steiner，伦敦），人们期待着对反应途径的分子理解。主要研究内容如下：（1）Hod和Hod突变蛋白对有机底物的解离常数的测定。(2)使用停流分光光度法/荧光分光光度法的预稳态动力学：检测光谱中间体并测定瞬态物质形成和衰减的速率常数。(3)Hod“突变蛋白”催化反应的动力学分析，以评估活性位点残基的作用。(4)分析pH值对动力学的影响，推导出中间物质和/或酶的可质子化基团的pKa值，并评估单个活性位点残基在质子转移步骤中的作用。(5)通过快速冷冻EPR补充UV/维斯停流实验检测和分析中间自由基，使用同位素替代底物详细分析淬灭自由基物种。

2-Methyl-3-hydroxy-4(1H)quinolone 2,4-dioxygenase (Hod) is a cofactor-independent dioxygenase with an α/β-hydrolase fold, catalyzing the cleavage of its organic substrate to carbon monoxide and N-acetylanthranilate. The overall goal of this project is to contribute to a comprehensive understanding of how cofactor-free oxygenases work, and to mechanistically compare cofactor-independent with metal- and flavin-dependent oxygenases. Transient kinetics studies will be performed to obtain insight into the time-dependent microscopic pathway of the Hod-catalyzed reaction. Combined with available structural data (Dr. R. Steiner, London), a molecular understanding of the reaction pathway is expected. The following issues will be addressed: (1) Determination of dissociation constants of Hod and Hod “muteins” for organic substrates. (2) Pre-steady state kinetics using stopped-flow spectrophotometry/spectrofluorometry: Detection of spectral intermediates and determination of rate constants for the formation and decay of transient species. (3) Kinetic analysis of the reaction catalyzed by Hod “muteins” to assess the role of active-site residues. (4) Analysis of the effects of pH on the kinetics, to deduce pKa values for intermediate species and/or for protonable groups of the enzyme, and to assess the role of individual active-site residues in proton transfer steps. (5) Detection and analysis of intermediate radicals by rapid-freeze EPR complementing the UV/Vis stopped-flow experiments, using isotope substituted substrates for detailed analysis of quenched radical species.