Spectroscopic, Structural and Kinetic Characterization of Sulfite Oxidase from Mu

描述（由申请人提供）：亚硫酸盐氧化酶（SO）是一种钼辅助因子依赖酶，催化亚硫酸盐氧化为硫酸盐，作为含硫氨基酸，半胱氨酸和蛋氨酸降解的最后一步。SO在生理上对人体代谢至关重要，遗传性亚硫酸盐氧化酶缺乏症会导致严重的神经损伤，其症状包括晶状体脱位、大脑发育迟缓、智力迟钝和过早死亡。SO缺乏要么是由于钼辅助因子合成的缺陷，要么是由于酶的各种点突变。虽然以前研究过来自多个来源的SO，但没有来自一个共同来源的全面的结构、光谱和反应性数据。本课题的主要研究目标是通过对易于在DL41 +大肠杆菌细胞中表达的小家鼠（Mus musculus）的SO进行一系列生物化学和生物物理研究，进一步了解SO的功能和机制以及点突变的影响。小鼠的SO与人类的酶有82%相同，但之前没有广泛的研究。研究小鼠SO突变体（变异体）性质的主要工具将是：1)激光闪光光解研究钼域和b型血红素域之间的分子内电子转移（IET）；2)高分辨率变频脉冲EPR实验，通过核耦合（1H、2H、31P、17O和33S）确定瞬态催化钼中心的结构；3)结晶和x射线结构测定作为pH和阴离子在介质中的函数。尽管在其他实验室进行了多次尝试，但完整的人类SO已被证明难以结晶。迄今为止，唯一完整的动物SO晶体结构是1997年野生型鸡SO的结构。对于人类SO，唯一可用的结构信息是分离的血红素结构域。来自拟南芥的植物SO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的动物系统，并且可以系统地产生大量的酶变体以供研究。小鼠SO符合这些标准，蛋白质表达的初步数据表明了所提出的研究的可行性。

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.