Wilson Heather L, Rajagopalan K V
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2004 Apr 9;279(15):15105-13. doi: 10.1074/jbc.M314288200. Epub 2004 Jan 16.
In the crystal structure of chicken sulfite oxidase, the residue Tyr(322) (Tyr(343) in human sulfite oxidase) was found to directly interact with a bound sulfate molecule and was proposed to have an important role in mediating the substrate specificity and catalytic activity of this molybdoprotein. In order to understand the role of this residue in the catalytic mechanism of sulfite oxidase, steady-state and stopped-flow analyses were performed on wild-type and Y343F human sulfite oxidase over the pH range 6-10. In steady-state assays of Y343F sulfite oxidase using cytochrome c as the electron acceptor, k(cat) was somewhat impaired ( approximately 34% wild-type activity at pH 8.5), whereas the K(m)(sulfite) showed a 5-fold increase over wild type. In rapid kinetic assays of the reductive half-reaction of wild-type human sulfite oxidase, k(red)(heme) changed very little over the entire pH range, with a significant increase in K(d)(sulfite) at high pH. The k(red)(heme) of the Y343F variant was significantly impaired across the entire pH range, and unlike the wild-type protein, both k(red)(heme) and K(d)(sulfite) were dependent on pH, with a significant increase in both kinetic parameters at high pH. Additionally, reduction of the molybdenum center by sulfite was directly measured for the first time in rapid reaction assays using sulfite oxidase lacking the N-terminal heme-containing domain. Reduction of the molybdenum center was quite fast (k(red)(Mo) = 972 s(-1) at pH 8.65 for wild-type protein), indicating that this is not the rate-limiting step in the catalytic cycle. Reduction of the molybdenum center of the Y343F variant by sulfite was more significantly impaired at high pH than at low pH. These results demonstrate that the Tyr(343) residue is important for both substrate binding and oxidation of sulfite by sulfite oxidase.
在鸡亚硫酸盐氧化酶的晶体结构中，发现残基Tyr(322)（人亚硫酸盐氧化酶中的Tyr(343)）与结合的硫酸根分子直接相互作用，并被认为在介导这种钼蛋白的底物特异性和催化活性方面具有重要作用。为了了解该残基在亚硫酸盐氧化酶催化机制中的作用，对野生型和Y343F人亚硫酸盐氧化酶在pH 6 - 10范围内进行了稳态和停流分析。在以细胞色素c作为电子受体的Y343F亚硫酸盐氧化酶的稳态测定中，k(cat)有所受损（在pH 8.5时约为野生型活性的34%），而K(m)(亚硫酸盐)比野生型增加了5倍。在野生型人亚硫酸盐氧化酶还原半反应的快速动力学测定中，k(red)(血红素)在整个pH范围内变化很小，在高pH时K(d)(亚硫酸盐)显著增加。Y343F变体的k(red)(血红素)在整个pH范围内均显著受损，与野生型蛋白不同，k(red)(血红素)和K(d)(亚硫酸盐)均依赖于pH，在高pH时两个动力学参数均显著增加。此外，在使用缺乏N端含血红素结构域的亚硫酸盐氧化酶的快速反应测定中，首次直接测量了亚硫酸盐对钼中心的还原。钼中心的还原相当快（野生型蛋白在pH 8.65时k(red)(Mo) = 972 s(-1)），表明这不是催化循环中的限速步骤。亚硫酸盐对Y343F变体钼中心的还原在高pH时比在低pH时受损更显著。这些结果表明，Tyr(343)残基对于亚硫酸盐氧化酶结合底物和氧化亚硫酸盐都很重要。
