Substrate Binding in Protein-tyrosine Phosphatase-like Inositol Polyphosphatases
Substrate Binding in Protein-tyrosine Phosphatase-like Inositol Polyphosphatases
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DOI：
10.1074/jbc.m111.309872
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发表时间：
2012-03-23
影响因子：
4.8
通讯作者：
Mosimann, Steven C.
中科院分区：
文献类型：
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作者：
Gruninger, Robert J.;Dobing, Selina;Mosimann, Steven C.
蛋白酪氨酸磷酸酶样肌醇多磷酸酶是微生物酶，其催化通过相对有序的途径从高度磷酸化的肌醇中逐步去除一种或多种磷酸盐。为了了解这些酶的底物特异性和动力学机制，我们已经确定了高分辨率，单晶，X-射线晶体结构的非活性反刍月形单胞菌PhyA的复合物与肌醇六磷酸和pentakisphosphate。这些结构提供了第一次瞥见的肌醇多磷酸酶-配体复合物符合其已知的特异性，并揭示了新的功能的动力学机制。为了补充结构研究，已经开发了荧光结合测定，并证明该酶的Kd比Km低几个数量级。连同快速动力学数据，这些结果表明，蛋白酪氨酸磷酸酶样肌醇多磷酸酶有两个步骤，底物结合机制，促进催化。
Protein-tyrosine phosphatase-like inositol polyphosphatases are microbial enzymes that catalyze the stepwise removal of one or more phosphates from highly phosphorylated myo-inositols via a relatively ordered pathway. To understand the substrate specificity and kinetic mechanism of these enzymes we have determined high resolution, single crystal, x-ray crystallographic structures of inactive Selenomonas ruminantium PhyA in complex with myo-inositol hexa-and pentakisphosphate. These structures provide the first glimpse of a myo-inositol polyphosphatase-ligand complex consistent with its known specificity and reveal novel features of the kinetic mechanism. To complement the structural studies, fluorescent binding assays have been developed and demonstrate that the K-d for this enzyme is several orders of magnitude lower than the K-m. Together with rapid kinetics data, these results suggest that the protein tyrosine phosphatase-like inositol polyphosphatases have a two-step, substrate-binding mechanism that facilitates catalysis.