Substrate specificity of the oncoprotein v-Fps: Site-specific mutagenesis of the putative P+1 pocket
Substrate specificity of the oncoprotein v-Fps: Site-specific mutagenesis of the putative P+1 pocket
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DOI：
10.1021/bi992096j
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发表时间：
2000-01-11
期刊：
影响因子：
2.9
通讯作者：
Adams, JA
中科院分区：
文献类型：
--
作者：
Konkol, L;Hirai, TJ;Adams, JA
基于胰岛素受体激酶的X射线结构[Hubbard，S. R.（1997）EMBO J. 16，5572-5581]，预测癌蛋白v-Fps（一种非受体酪氨酸蛋白激酶）中的Arg-1130与底物肽中的P+1谷氨酸相互作用。为了确定该残基是否是v-Fps中的重要识别元件，将Arg-1130用亮氨酸（R1130 L）和谷氨酸（R1130 E）取代。评估这些突变体磷酸化肽EAEIYXAIE（其中X是谷氨酸、丙氨酸或赖氨酸）的能力。在限制底物浓度（即，k（cat）/K-m条件）表明底物特异性被P+1口袋的静电环境改变。当口袋显示正电荷（Arg-1130;野生型）、不带电荷（R1130 L）或负电荷（R1130 E）时，v-Fps优选以200：1、9：1或1：1的比例磷酸化谷氨酸肽而不是赖氨酸肽。虽然谷氨酸肽的k（cat）/K-m对于野生型比R1130 E高50倍，但是赖氨酸肽的k（cat）/K-m对于R1130 E比野生型高3倍，相对底物特异性变化150倍。使用粘度技术分析动力学机制中的各个步骤表明，野生型酶与谷氨酸肽的结合比丙氨酸肽好S倍，并且比赖氨酸肽好至少10倍。对于R1130 L，该裕度范围显著减小，并且对于R1130 E，未观察到结合偏好。尽管如此，赖氨酸肽与R1130 E的结合比与野生型的结合好至少4倍，而谷氨酸肽与R1130 E的结合比与野生型的结合差3倍。对于三种肽，突变体使磷酰基转移速率降低4-30倍，表明Arg-1130有助于定位酪氨酸以获得最佳催化。数据表明，v-Fps中的单个突变可以显著改变相对底物特异性约2个数量级，其中至少50%的这种效应通过肽结合亲和力的相对变化发生。
Based on the X-ray structure of the insulin receptor kinase [Hubbard, S. R. (1997) EMBO J. 16, 5572-5581], Arg-1130 in the oncoprotein v-Fps, a nonreceptor tyrosine protein kinase, is predicted to interact with the P+1 glutamate in substrate peptides. To determine whether this residue is an important recognition element in v-Fps, Arg-1130 was substituted with leucine (R1130L) and glutamic acid (R1130E). The ability of these mutants to phosphorylate the peptide EAEIYXAIE, where X is glutamic acid, alanine, or lysine, was assessed. A comparison of the rates of peptide phosphorylation under limiting substrate concentrations (i.e., k(cat)/K-m conditions) indicates that substrate specificity is altered by the electrostatic environment of the P+1 pocket. When the pocket displays a positive charge (Arg-1130; wild type), no charge (R1130L), or a negative charge (R1130E), v-Fps prefers to phosphorylate the glutamate peptide over the lysine peptide by a 200:1, 9:1, or 1:1 margin. While k(cat)/K-m for the glutamate peptide is 50-fold higher for wild type compared to R1130E, k(cat)/K-m for the lysine peptide is 3-fold higher for R1130E compared to wild type, a 150-fold change in relative substrate specificity. Analysis of the individual steps in the kinetic mechanism using viscosometric techniques indicates that the wild-type enzyme binds the glutamate peptide S-fold better than the alanine peptide and, at least, 10-fold better than the lysine peptide. For R1130L, this margin range is reduced substantially, and for R1130E, no binding preference is observed. Nonetheless, the lysine peptide binds, at least, 4-fold better to R1130E than to wild type, and the glutamate peptide binds 3-fold poorer to R1130E than to wild type. The mutants lower the phosphoryl transfer rate by 4-30-fold for the three peptides, suggesting that Arg-1130 helps to position the tyrosine for optimum catalysis. The data indicate that a single mutation in v-Fps can alter significantly the relative substrate specificity by about 2 orders of magnitude with, at least, 50% of this effect occurring through relative changes in peptide binding affinity.