Feng Changjian, Wilson Heather L, Hurley John K, Hazzard James T, Tollin Gordon, Rajagopalan K V, Enemark John H
Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA.
Biochemistry. 2003 Oct 28;42(42):12235-42. doi: 10.1021/bi0350194.
Arginine 160 in human sulfite oxidase (SO) is conserved in all SO species sequenced to date. Previous steady-state kinetic studies of the R160Q human SO mutant showed a remarkable decrease in k(cat)/K(m)(sulfite) of nearly 1000-fold, which suggests that Arg 160 in human SO makes an important contribution to the binding of sulfite near the molybdenum cofactor [Garrett, R. M., Johnson, J. L., Graf, T. N., Feigenbaum, A., Rajagopalan, K. V. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6394-6398]. In the crystal structure of chicken SO, Arg 138, the equivalent of Arg 160 in human SO, is involved in the formation of a positively charged sulfite binding site [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garnett, R. M., Rajagopalan, K. V., Enemark, J. H., Rees, D. C. (1997) Cell 91, 973-983]. To further assess the role of Arg 160 in human SO, intramolecular electron transfer (IET) rates between the reduced heme [Fe(II)] and oxidized molybdenum [Mo(VI)] centers in the wild type, R160Q, and R160K human SO forms were investigated by laser flash photolysis. In the R160Q mutant, the IET rate constant at pH 6.0 was decreased by nearly 3 orders of magnitude relative to wild type, which indicates that the positive charge of Arg 160 is essential for efficient IET in human SO. Furthermore, the IET rate constant for the R160K mutant is about one-fourth that of the wild type enzyme, which strongly indicates that it is the loss of charge of Arg 160, and not its precise location, that is responsible for the much larger decrease in IET rates in the R160Q mutant. Steady-state kinetic measurements indicate that IET is rate-limiting in the catalytic cycle of the R160Q mutant. Thus, the large decrease in the IET rate constant rationalizes the fatal impact of this mutation in patients with this genetic disorder.
人亚硫酸盐氧化酶（SO）中的精氨酸160在迄今为止测序的所有SO物种中都是保守的。先前对R160Q人SO突变体的稳态动力学研究表明，k(cat)/K(m)(亚硫酸盐)显著降低了近1000倍，这表明人SO中的精氨酸160对亚硫酸盐在钼辅因子附近的结合有重要贡献[加勒特，R.M.，约翰逊，J.L.，格拉夫，T.N.，费根鲍姆，A.，拉贾戈帕兰，K.V.（1998年）《美国国家科学院院刊》95，6394 - 6398]。在鸡SO的晶体结构中，精氨酸138（相当于人SO中的精氨酸160）参与形成一个带正电的亚硫酸盐结合位点[基斯克，C.，欣德林，H.，帕切科，A.，韦赫比，W.，加内特，R.M.，拉贾戈帕兰，K.V.，埃内马克，J.H.，里斯，D.C.（1997年）《细胞》91，973 - 983]。为了进一步评估精氨酸160在人SO中的作用，通过激光闪光光解研究了野生型、R160Q和R160K人SO形式中还原型血红素[Fe(II)]和氧化型钼[Mo(VI)]中心之间的分子内电子转移（IET）速率。在R160Q突变体中，pH 6.0时的IET速率常数相对于野生型降低了近3个数量级，这表明精氨酸160的正电荷对于人SO中高效的IET至关重要。此外，R160K突变体的IET速率常数约为野生型酶的四分之一，这强烈表明是精氨酸160电荷的丧失，而非其精确位置，导致了R160Q突变体中IET速率的大幅下降。稳态动力学测量表明，IET在R160Q突变体的催化循环中是限速步骤。因此，IET速率常数的大幅下降解释了这种突变对患有这种遗传疾病患者的致命影响。
