Functions of the proteins of the sulfur-oxidizing enzyme system of Paracoccus pantotrophus

该项目的目的是确定参与泛养副球菌硫氧化（Sox）酶系统的每种蛋白质的实际反应。该酶系由SoxYZ、SoxXA、SoxB和SoxCD四种蛋白质组成。现在SoxYZ可以被活化以产生均匀的高比活性，这是研究Sox酶系统的催化特性的基础。(i)Sox系统的动力学将是独特的，因为没有其他已知的酶系统形成多个瞬时复合物以将一种底物转化为一种产物。(ii)将分析单个Sox蛋白的特定反应，这需要SoxY负载不同氧化态的硫原子。此外，（iii）将通过衰减全反射分析和等温量热法检查Sox蛋白之间的特异性蛋白质-蛋白质相互作用。(iv)活化的SoxYZ蛋白的主要成分是通过蛋白间二硫键连接的SoxY-Y同源二聚体。将检查蛋白间二硫键对反应的意义。(v)SoxZ是SoxY的复合物伴侣，对SoxY的活性至关重要，但其在SoxY的构象转换中的作用、其反应性或SoxYZ复合物的稳定性尚不清楚，将通过动力学和定点突变来分析。

The objective of the project is the determination of the actual reaction of each protein participating in the sulfur-oxidizing (Sox) enzyme system of Paracoccus pantotrophus. This enzyme system is composed of four proteins, SoxYZ, SoxXA, SoxB and SoxCD. SoxYZ can now be activated to yield a uniforn high specific activity which is the basis to examine the catalytic characteristics of the Sox enzyme system. (i) Kinetics of the Sox system will be be unique as no other known enzyme system forms multiple transient complexes to convert one substrate to one product. (ii) Specific reactions of the individual Sox proteins will be analyzed which requires the loading of SoxY with sulfur atoms of different oxidation states. In addition (iii) specific protein-protein interactions among the Sox proteins will be examined by attenuated total reflection analysis and isothermal calorimetry. (iv) The major component of activated SoxYZ protein is the SoxY-Y homodimer linked by an interprotein disulfide. The significance of the interprotein disulfide for the reaction will be examined. (v) SoxZ, the complex partner of SoxY, is essential for activity of SoxY but its role in conformational switch of SoxY, its reactivity or stabilization of the SoxYZ complex is unknown and will be analyzed by kinetics and site-directed mutagenesis.