Spectroscopic, Structural and Kinetic Characterization of Sulfite Oxidase from Mu

说明(申请人提供)：亚硫酸盐氧化酶(SO)是一种钼辅酶，催化亚硫酸盐氧化成硫酸盐，作为含硫氨基酸、半胱氨酸和蛋氨酸降解的最后一步。在人体新陈代谢中，生理上也是至关重要的，遗传性亚硫酸盐氧化酶缺乏症会导致严重的神经损伤，症状包括眼晶状体脱位、大脑发育减弱、智力低下和过早死亡。因此，缺乏钼的原因要么是钼辅助因子合成的缺陷，要么是该酶的各种点突变所致。虽然以前已经研究了来自几个来源的SO，但还没有来自同一来源的全面的结构、光谱和反应性数据。这项研究的主要目标是通过一系列整合的生化和生物物理研究来深入了解SO的功能和机制，以及点突变的影响。老鼠的SO与人类的酶有82%的同源性，但之前还没有得到广泛的研究。研究小鼠SO突变体(变异体)的性能的主要工具将是：1)激光闪光光解研究钼结构域和b型血红素结构域之间的分子内电子转移(IET)；2)高分辨率变频脉冲EPR实验通过核偶联(1H，2H，31P，17O和33S)确定瞬时催化钼中心的结构；3)结晶和X射线结构测定随介质中pH和阴离子的变化。尽管在其他实验室进行了多次尝试，但事实证明，完整的人类SO很难结晶。到目前为止，完整动物的晶体结构是唯一的，1997年野生鸡的结构也是如此。对于人类SO，唯一可用的结构信息是孤立的血红素结构域。植物SO，来自拟南芥，只有一个钼结构域。因此，需要一种与人类SO具有高度序列同源性并且可以系统地产生大量酶变体用于研究的动物系统。小鼠So符合这些标准，表达该蛋白的初步数据表明了拟议研究的可行性。

DESCRIPTION (provided by applicant): Sulfite Oxidase (SO) is a molybdenum cofactor dependent enzyme that catalyzes the oxidation of sulfite to sulfate as the final step in the degradation of the sulfur-containing amino acids, cysteine and methionine. SO is physiologically vital in human metabolism, and hereditary sulfite oxidase deficiency results in severe neurological damage with symptoms that include dislocation of the ocular lenses, attenuated growth of the brain, mental retardation and early death. SO deficiency results either from a defect in the synthesis of the molybdenum cofactor or from various point mutations with the enzyme. While SO's from several sources have been previously studied, there is no comprehensive set of structural, spectroscopic and reactivity data from a common source. The primary goal of the research presented in this proposal is to gain greater insight into the function and mechanism of SO and the effect of point mutations through an integrated series of biochemical and biophysical studies of SO from Mus musculus (house mouse), which is readily expressed in DL41 plus E. coli cells. Mouse SO is 82% identical with the human enzyme, but has not been previously investigated extensively. The primary tools for investigating the properties of mutants (variants) of mouse SO will be: 1) Laser flash photolysis studies of intramolecular electron transfer (IET) between the molybdenum domain and the b type heme domain; 2) high resolution variable frequency pulsed EPR experiments to determine the structure of the transient catalytic molybdenum center through nuclear couplings (1H, 2H, 31P, 17O and 33S); 3) crystallization and X-ray structure determination as a function of pH and anions in the media. Despite numerous attempts in other laboratories, intact human SO has proven difficult to crystallize. To date the only crystal structure for intact animal SO is the 1997 structure of wild-type chicken SO. For human SO, the only available structural information is for the isolated heme domain. Plant SO, from Arabidopsis thaliana only has a molybdenum domain. Thus, there is a need for an animal system that has a high sequence homology to human SO and for which substantial quantities of enzyme variants can be systematically produced for study. Mouse SO meets these criteria and preliminary data on expression of the protein show the feasibility of the proposed studies.