Kinetic and mutational analyses of the regulation of phosphoribulokinase by thioredoxins
Kinetic and mutational analyses of the regulation of phosphoribulokinase by thioredoxins
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DOI：
10.1074/jbc.m001936200
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发表时间：
2000-06-16
影响因子：
4.8
通讯作者：
Hartman, FC
中科院分区：
文献类型：
--
作者：
Geck, MK;Hartman, FC
尽管很少有支持性的数据，差异靶蛋白的敏感性，氧化还原调节硫氧还蛋白（Trx）fend Trx在已被调用占两个不同的Trx在叶绿体。然而，这一假设还没有得到严格的测试与磷酸核酮糖激酶（磷酸核酮糖激酶），一个支点的氧化还原调节的卡尔文循环。Trx研究的先决条件是，检查二硫苏糖醇、2-巯基乙醇和谷胱甘肽对菠菜淀粉酶的活化。与之前的报道相反，每种都激活了PRK，但只有二硫苏糖醇支持Trx依赖性激活。活化的比较动力学表明，Trx m比Trx f更有效，因为其V-max高40%，但S-0.5相似。激活是不敏感的核酮糖二磷酸羧化酶，这可能是复杂的在体内。为了探索的基础上优越的Trx m，我们的特点是定点突变的Trx f，其中独特的残基在保守区域被替换与Trx m对应或删除。这些变化通常导致V-max增强，其中最大的（6倍）发生在T105 I，反映了与活性位点相反的疏水区域中的取代。包括本研究在内，几种不同Trx调节酶的活化动力学表明叶绿体Trx功能的冗余。
Despite little supportive data, differential target protein susceptibility to redox regulation by thioredoxin (Trx) fend Trx in has been invoked to account for two distinct Trxs in chloroplasts. However, this postulate has not been rigorously tested with phosphoribulokinase (PRK), a fulcrum for redox regulation of the Calvin cycle. Prerequisite to Trx studies, the activation of spinach PRK by dithiothreitol, 2-mercaptoethanol, and glutathione was examined. Contrary to prior reports, each activated PRK, but only dithiothreitol supported Trx-dependent activation. Comparative kinetics of activation of PRK showed Trx m to be more efficient than Trx f because of its 40% higher V-max but similar S-0.5. Activations were insensitive to ribulosebisphosphate carboxylase, which may complex with PRK in vivo. To probe the basis for superiority of Trx m, we characterized site-directed mutants of Trx f, in which unique residues in conserved regions were replaced with Trx m counterparts or deleted. These changes generally resulted in V-max enhancements, the largest (6-fold) of which occurred with T105I, reflective of substitution in a hydrophobic region that opposes the active site. inclusive of the present study activation kinetics of several different Trx-regulated enzymes indicate redundancy in the functions of the chloroplastic Trxs.