Altering the Nucleophile Specificity of a Protein-tyrosine Phosphatase-catalyzed Reaction
Altering the Nucleophile Specificity of a Protein-tyrosine Phosphatase-catalyzed Reaction
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DOI：
10.1074/jbc.273.10.5484
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发表时间：
1998-03
期刊：
影响因子：
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通讯作者：
Y. Zhao;Li Wu;S. Noh;K. Guan;Zhon-Yin Zhang
中科院分区：
文献类型：
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作者：
Y. Zhao;Li Wu;S. Noh;K. Guan;Zhon-Yin Zhang
蛋白酪氨酸磷酸酶（PTPases）催化涉及半胱氨酸磷酸中间体，其中磷酸基不能转移到除水以外的亲核试剂上。双特异性磷酸酶和低分子量磷酸酶利用相同的化学机制进行催化，并含有与PTPases相同的（H/V）C(X)5R（S/T）特征基序。有趣的是，后两组磷酸酶除了催化水外，还能催化磷酸酰基转化为醇。PTPase家族独有的两个不变的Gln残基位于活性位点。Gln-446（以及较小程度的Gln-450）到Ala， Asn或Met（但不包括Glu）残基的突变破坏了Gln-446侧链和亲核水之间的分叉氢键，并赋予了磷酸化酶活性。因此，保守的Gln-446残基负责维持PTPases严格的水解活性，并防止PTPases作为激酶磷酸化不需要的底物。这就解释了为什么PTPases中磷酸化酶中间体的磷酰转移只能发生在水上，而不能发生在其他亲核受体上。详细的动力学分析也表明了Gln-446和Gln-450在PTPase催化中的作用。虽然Gln-446在磷酸酶的形成步骤中不是必需的，但它在中间体的水解过程中发挥了重要作用，它将亲核水隔离并定位在活性位点上，以便对半胱氨酸磷酸中间体的磷原子进行在线攻击。Gln-450通过结合水分子与磷基部分相互作用，可能对活性位点残基的精确定位起作用，这对于底物结合和过渡态稳定在这两个化学步骤中都是重要的。
Protein-tyrosine phosphatases (PTPases) catalysis involves a cysteinyl phosphate intermediate, in which the phosphoryl group cannot be transferred to nucleophiles other than water. The dual specificity phosphatases and the low molecular weight phosphatases utilize the same chemical mechanism for catalysis and contain the same (H/V)C(X)5R(S/T) signature motif present in PTPases. Interestingly, the latter two groups of phosphatases do catalyze phosphoryl transfers to alcohols in addition to water. Unique to the PTPase family are two invariant Gln residues which are located at the active site. Mutations at Gln-446 (and to a much smaller extent Gln-450) to Ala, Asn, or Met (but not Glu) residues disrupt a bifurcated hydrogen bond between the side chain of Gln-446 and the nucleophilic water and confer phosphotransferase activity to theYersinia PTPase. Thus, the conserved Gln-446 residue is responsible for maintaining PTPases’ strict hydrolytic activity and for preventing the PTPases from acting as kinases to phosphorylate undesirable substrates. This explains why phosphoryl transfer from the phosphoenzyme intermediate in PTPases can only occur to water and not to other nucleophilic acceptors. Detailed kinetic analyses also suggest roles for Gln-446 and Gln-450 in PTPase catalysis. Although Gln-446 is not essential for the phosphoenzyme formation step, it plays an important role during the hydrolysis of the intermediate by sequestering and positioning the nucleophilic water in the active site for an in-line attack on the phosphorus atom of the cysteinyl phosphate intermediate. Gln-450 interacts through a bound water molecule with the phosphoryl moiety and may play a role for the precise alignment of active site residues, which are important for substrate binding and transition state stabilization for both of the chemical steps.