Probing the catalytic mechanism of the insulin receptor kinase with a tetrafluorotyrosine-containing peptide substrate
Probing the catalytic mechanism of the insulin receptor kinase with a tetrafluorotyrosine-containing peptide substrate
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DOI：
10.1074/jbc.m003524200
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发表时间：
2000-09-29
影响因子：
4.8
通讯作者：
Kohanski, RA
中科院分区：
文献类型：
--
作者：
Ablooglu, AJ;Till, JH;Kohanski, RA
通过稳态动力学和 X 射线晶体学研究了合成四氟酪氨酰肽底物与胰岛素受体的激活酪氨酸激酶结构域的相互作用。 pH 速率曲线表明，酶催化的磷酸化需要中性苯酚，而不是化学反应性更强的酚盐离子。与溶液相比，酶上四氟酪氨酰羟基的 pK(α) 升高 2 个 pH 单位，而酚盐阴离子则充当酪氨酸激酶的弱竞争性抑制剂。二元酶-底物复合物的结构显示，四氟酪氨酰基 OH 基团位于距 Asp(1132) 和 Arg(1136) 侧链的氢键距离处，与 pK(α) 的升高一致。这些发现强烈支持有利于解离过渡态的反应机制。
The interaction of a synthetic tetrafluorotyrosyl peptide substrate with the activated tyrosine kinase domain of the insulin receptor was studied by steady-state kinetics and x-ray crystallography. The pH-rate profiles indicate that the neutral phenol, rather than the chemically more reactive phenoxide ion, is required for enzyme-catalyzed phosphorylation. The pK(alpha) of the tetrafluorotyrosyl hydroxyl is elevated 2 pH units on the enzyme compared with solution, whereas the phenoxide anion species behaves as a weak competitive inhibitor of the tyrosine kinase. A structure of the binary enzyme-substrate complex shows the tetrafluorotyrosyl OH group at hydrogen bonding distances from the side chains of Asp(1132) and Arg(1136), consistent with elevation of the pK(alpha). These findings strongly support a reaction mechanism favoring a dissociative transition state.