Effect of ionic strength and oxidation on the P-loop conformation of the protein tyrosine phosphatase-like phytase, PhyAsr
Effect of ionic strength and oxidation on the P-loop conformation of the protein tyrosine phosphatase-like phytase, PhyAsr
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DOI：
10.1111/j.1742-4658.2008.06524.x
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发表时间：
2008-08-01
期刊：
影响因子：
5.4
通讯作者：
Mosimann, Steven C.
中科院分区：
文献类型：
--
作者：
Gruninger, Robert J.;Selinger, L. Brent;Mosimann, Steven C.
来自反刍硒单胞菌的蛋白酪氨酸磷酸酶（PTP）样phytase （PhyAsr）是PTP超家族的新成员，也是唯一被描述的水解肌醇-1,2,3,4,5,6-己基磷酸的成员。除了PhyAsr独特的底物特异性外，据报道，当配体在低离子强度下结合时，磷酸盐结合环（p环）的构象从开放（非活性）转变为封闭（活性）。在高离子强度下，p环在有配体和没有配体的情况下都呈封闭的活性构象。为了验证离子强度的变化是否能诱导p环运动，我们考察了离子强度对PhyAsr催化效率的影响，并测定了几种离子强度下酶的结构。PhyAsr的催化效率对离子强度高度敏感，当离子强度从100 mM增加到500 mM时，k(cat)/ k -m增加了7倍，k -m减少了9倍。令人惊讶的是，尽管没有配体，但在所有离子强度下都观察到p环具有催化能力的构象。在这里，我们提供了结构证据，证明PhyAsr的离子强度依赖性和p环的构象变化没有联系。此外，我们证明了先前报道的p环构象变化是活性位点硫代酸酯不可逆氧化的结果。最后，我们对在所有氧化PTP结构中观察到的p环构象变化进行了合理化。
The protein tyrosine phosphatase (PTP)-like phytase, PhyAsr, from Selenomonas ruminantium is a novel member of the PTP superfamily, and the only described member that hydrolyzes myo-inositol-1,2,3,4,5,6-hexakisphosphate. In addition to the unique substrate specificity of PhyAsr, the phosphate-binding loop (P-loop) has been reported to undergo a conformational change from an open (inactive) to a closed (active) conformation upon ligand binding at low ionic strength. At high ionic strengths, the P-loop was observed in the closed, active conformation in both the presence and absence of ligand. To test whether the P-loop movement can be induced by changes in ionic strength, we examined the effect that ionic strength has on the catalytic efficiency of PhyAsr, and determined the structure of the enzyme at several ionic strengths. The catalytic efficiency of PhyAsr is highly sensitive to ionic strength, with a seven-fold increase in k(cat)/K-m and a ninefold decrease in K-m when the ionic strength is increased from 100 to 500 mM. Surprisingly, the P-loop is observed in the catalytically competent conformation at all ionic strengths, despite the absence of a ligand. Here we provide structural evidence that the ionic strength dependence of PhyAsr and the conformational change in the P-loop are not linked. Furthermore, we demonstrate that the previously reported P-loop conformational change is a result of irreversible oxidation of the active site thiolate. Finally, we rationalize the observed P-loop conformational changes observed in all oxidized PTP structures.