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（Hod）是一种不依赖于辅酶的双加氧酶，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 -乙酰氨基苯甲酸酯。该项目的总体目标是全面了解无辅助因子加氧酶的工作原理，并从机制上比较不依赖辅助因子的加氧酶与金属和黄素依赖的加氧酶。瞬态动力学研究将被执行，以获得洞察时间依赖的微观途径的催化反应。结合现有的结构数据（Dr. R. Steiner, London），对反应途径的分子理解有望实现。以下问题将被解决：(1)确定有机底物的Hod和Hod“突变蛋白”的解离常数。(2)使用止流分光光度法/荧光光谱法的预稳态动力学：检测光谱中间体和测定瞬态物质形成和衰变的速率常数。(3) Hod“muteins”催化反应动力学分析，评价活性位点残基的作用。(4)分析pH对动力学的影响，推断酶的中间物质和/或可质子基团的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)利用速冻EPR技术对中间自由基进行检测和分析，并与UV/Vis停流实验相结合，利用同位素取代底物对淬灭自由基进行详细分析。

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.