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)是一种非依赖辅因子的双加氧酶，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-乙酰邻氨基苯甲酸。这个项目的总体目标是有助于全面了解无辅因子加氧酶的工作原理，并从机械上比较辅因子非依赖加氧酶与金属和黄素依赖加氧酶的作用。将进行瞬时动力学研究，以深入了解HOD催化反应的随时间变化的微观途径。结合现有的结构数据(R.Steiner博士，伦敦)，有望从分子上了解反应途径。将解决以下问题：(1)有机底物Hod和Hod“突变体”解离常数的测定。(2)用停流分光光度/荧光分光光度法测定稳态前动力学：检测光谱中间体和测定瞬时物种的形成和衰变的速率常数。(3)对Hod“突变体”催化的反应进行动力学分析，以评估活性中心残基的作用。(4)分析pH对动力学的影响，推算酶中间物种和/或可质子化基团的pKa值，并评估单个活性中心残基在质子转移步骤中的作用。(5)快速冷冻EPR检测和分析中间自由基，补充UV/VIS停流实验，使用同位素替代底物对猝灭的自由基物种进行详细分析。

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.