Feng Changjian, Wilson Heather L, Tollin Gordon, Astashkin Andrei V, Hazzard James T, Rajagopalan K V, Enemark John H
Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA.
Biochemistry. 2005 Oct 25;44(42):13734-43. doi: 10.1021/bi050907f.
Mutations G473D and A208D were identified in patients with isolated sulfite oxidase (SO) deficiency, and the equivalent amino acids (G451 and A186, respectively) have been localized to the vicinity of the molybdopterin active site in the X-ray structure of chicken SO [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garrett, R. M., Rajagopalan, K. V., Enemark, J. H., and Rees, D. C. (1997) Cell 91, 973-983]. To assess the effects of these mutations in human SO, steady-state kinetic studies of enzyme turnover and laser flash photolysis measurements of intramolecular electron transfer (IET) rate constants between the reduced heme [Fe(II)] and Mo(VI) centers were carried out in the recombinant G473D, G473A, G473W, G473D/R212A, and A208D human SO mutants. In the G473D and A208D mutants, the IET rate constants at pH 6.0 are decreased by 3 orders of magnitude relative to that of the wild type. Steady-state kinetic measurements indicate that the IET process is the rate-limiting step in the catalytic cycle of these two mutants. Thus, the large decreases in the IET rate constants and the kcat values, and the large increases in the Km(sulfite) values, rationalize the fatal impact of these mutations. Far-UV CD spectra of G473D indicate that the protein backbone conformation is remarkably changed, and the sedimentation equilibrium indicates that the protein is monomeric. Furthermore, EPR studies also suggest that the active site structure of the Mo(V) form of A208D is different from that of the wild type. In contrast, similar studies on G473A show that it is dimeric, that its Mo(V) active site structure is similar to that of the wild type, and that its IET rate constant is only 2.6-fold smaller than that of the wild type. IET in G473W is severely impaired, and no IET is observed for G473D/R212A. In chicken SO, the equivalent residues (G451 and A186) are both buried inside the protein. Thus, for human SO, the mutations to charged residues at the equivalent sites most likely cause crucial global or localized structural changes, and expose an alternative docking site that may compete with the Mo domain for docking of the heme, thereby retarding IET and efficient catalytic turnover of the sulfite oxidation reaction.
在孤立性亚硫酸盐氧化酶（SO）缺乏症患者中鉴定出了G473D和A208D突变，鸡SO的X射线结构中，相应的氨基酸（分别为G451和A186）已定位到钼蝶呤活性位点附近[基斯克尔，C.，欣德林，H.，帕切科，A.，韦赫比，W.，加勒特，R.M.，拉贾戈帕兰，K.V.，埃内马克，J.H.，和里斯，D.C.（1997年）《细胞》91卷，973 - 983页]。为了评估这些突变对人SO的影响，对重组G473D、G473A、G473W、G473D/R212A和A208D人SO突变体进行了酶周转的稳态动力学研究以及还原血红素[Fe(II)]与Mo(VI)中心之间分子内电子转移（IET）速率常数的激光闪光光解测量。在G473D和A208D突变体中，pH 6.0时的IET速率常数相对于野生型降低了3个数量级。稳态动力学测量表明，IET过程是这两个突变体催化循环中的限速步骤。因此，IET速率常数和kcat值的大幅降低以及Km（亚硫酸盐）值的大幅升高，解释了这些突变的致命影响。G473D的远紫外圆二色光谱表明蛋白质主链构象发生了显著变化，沉降平衡表明该蛋白质是单体。此外，电子顺磁共振研究还表明，A208D的Mo(V)形式的活性位点结构与野生型不同。相比之下，对G473A的类似研究表明它是二聚体，其Mo(V)活性位点结构与野生型相似，其IET速率常数仅比野生型小2.6倍。G473W中的IET严重受损，G473D/R212A未观察到IET。在鸡SO中，相应的残基（G451和A186）都埋在蛋白质内部。因此，对于人SO，在等效位点突变为带电荷的残基很可能会导致关键的全局或局部结构变化，并暴露出一个可能与Mo结构域竞争血红素对接的替代对接位点，从而阻碍IET和亚硫酸盐氧化反应的有效催化周转。
