Kinetic analysis of a protein tyrosine kinase reaction transition state in the forward and reverse directions
Kinetic analysis of a protein tyrosine kinase reaction transition state in the forward and reverse directions
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DOI：
10.1021/ja9808393
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发表时间：
1998-07-22
影响因子：
15
通讯作者：
Cole, PA
中科院分区：
文献类型：
--
作者：
Kim, K;Cole, PA
蛋白酪氨酸激酶催化γ-磷酰基从ATP转移到蛋白质中的酪氨酸残基，并且是细胞信号转导中的重要酶。通过分析一系列含氟酪氨酸的肽底物，研究了Csk催化蛋白酪氨酸激酶反应的催化磷酰基转移过渡态。对于五种这样的含氟酪氨酸的肽底物，确定了酪氨酸类似物苯酚PK（a）和负责pH速率曲线分析的基本分支的可电离基团之间存在良好的一致性。这表明底物酪氨酸酚必须是中性的才有酶活性。与以前的数据表明一个小的β（亲核）系数（0-0.1），这些结果强烈支持解离过渡态的磷酰基转移。此外，测量反向蛋白酪氨酸激酶反应的β（离去基团）系数，结果显示为-0.3。这个值是在良好的协议与先前报道的非酶模型磷酰基转移反应进行酸性条件下（pH 4），是最容易解释的过渡态与显着的质子转移到离去的苯酚。
Protein tyrosine kinases catalyze the transfer of the gamma-phosphoryl,group from ATP to tyrosine residues in proteins and are important enzymes in cell signal transduction. We have investigated the catalytic phosphoryl transfer transition state of a protein tyrosine kinase reaction catalyzed by Csk by analyzing a series of fluorotyrosine-containing peptide substrates. It was established for five such fluorotyrosine-containing peptide substrates that there is good agreement between the tyrosine analogue phenol pK(a) and the ionizable group responsible for the basic limb of a pH rate profile analysis. This indicates that the substrate tyrosine phenol must be neutral to be enzymatically active. Taken together with previous data indicating a small beta(nucleophile) coefficient (0-0.1), these results strongly support a dissociative transition state for phosphoryl transfer. In addition, the beta(leaving group) coefficient was measured for the reverse protein tyrosine kinase reaction and shown to be -0.3. This value is in good agreement with a previously reported nonenzymatic model phosphoryl transfer reaction carried out under acidic conditions (pH 4) and is most readily explained by a transition state with significant proton transfer to the departing phenol.