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

加氧酶的生物化学：属于α/α-水解酶折叠家族的不依赖辅因子、CO 形成的双加氧酶的机制研究

基本信息

批准号：
91767900
负责人：
Professorin Dr. Susanne Fetzner
金额：
--
依托单位：
Institut für Molekulare Mikrobiologie und Biotechnologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2008
资助国家：
德国
起止时间：
2007-12-31 至 2013-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/91767900?language=en
关键词：
Biochemistry oxygenases Mechanistic studies cofactor

项目摘要

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.

2-甲基-3-羟基-4（1H）奎诺酮2,4-二氧酶（HOD）是一种辅助因子独立的二氧酶，具有α/β-氢化酶折叠，催化其有机底物对碳单氧一和非乙酰基苯基苯基丙烷的裂解。该项目的总体目标是有助于全面了解无辅助因子的氧合酶的工作方式，并将机械地与金属和黄素依赖性的氧酶进行机械比较。将进行瞬时动力学研究，以了解HOD催化反应的时间依赖性微观途径。结合可用的结构数据（R. Steiner博士，伦敦），对反应途径的分子了解。将解决以下问题：（1）确定有机底物的HOD和HOD“静标素”的解离常数。（2）使用停止流量分光光度法/光谱荧光测定法的前态态动力学：检测光谱中间体并确定瞬时物种形成和衰减的速率常数。（3）对用hod“静s”催化的反应的动力学分析，以评估活跃位点残差的作用。（4）分析pH对动力学的作用，推断中间物种和/或酶的质子基团的PKA值，并评估单个活性位点残差在质子传递步骤中的作用。（5）使用同位素取代的底物来详细分析淬灭自由基物种的详细分析，通过快速冻结EPR完成UV/VIS止流实验来检测和分析中间自由基。