Insight into tyrosine phosphorylation in v-Fps using proton inventory techniques
Insight into tyrosine phosphorylation in v-Fps using proton inventory techniques
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DOI：
10.1021/bi960613h
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发表时间：
1996-08-20
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：
Adams, JA
利用质子存量和粘度技术分析了非受体酪氨酸蛋白激酶v-Fps的激酶结构域中的磷酰基转移步骤。后者的研究表明，两个九个残基的肽底物的v-Fps，肽I（EAEAYEAIE）和II（EAEIYEAIE），是在快速平衡的活性位点，并结合类似的亲和力（K-S = 2.2和1.7毫米的肽I和II）。虽然磷酰基转移是在中性pH下肽I（5 s（-1））的k（cat）中的限速步骤，但肽II通过动力学机制转化为产物，其中磷酰基转移（45 s（-1））和产物释放（20 s（-1））部分控制该参数。在95%D_2O中，两种多肽的磷酸化反应均存在明显的溶剂同位素效应（k（0）/k（n）约为1.6）。肽I的K（cat）上的质子库存是线性的，表明磷酰基转移步骤与单个质子转移相关。相反，质子库存k（猫）的肽II是“弓”了，符合一个“虚拟”的过渡状态，其中磷酰基转移和产品释放步骤部分控制这个参数。两种肽的k（cat）/K-肽上缺乏溶剂同位素效应可以通过对底物结合的平衡同位素效应来解释，该效应抵消了磷酰基转移的动力学同位素效应。与该提议一致，抑制剂肽EAEIFEAIE的K-I在0和60%D2O中分别为11 +/- 0.08和6.5 +/- 0.82 mM。使用修改形式的格罗斯-巴特勒方程的质子库存图拟合提供肽I和II的1.7和3.6的固有同位素效应。合并后的数据是一致的机制，涉及酸-碱催化剂或构象变化之前的产品，伴随着一个单一的氢供体-受体对的破坏释放。
The phosphoryl group transfer step in the kinase domain of v-Fps, a nonreceptor tyrosine protein kinase, was analyzed using proton inventory and viscosometric techniques. The latter studies show that two nine-residue peptide substrates for v-Fps, peptides I (EAEAYEAIE) and II (EAEIYEAIE), are in rapid equilibrium with the active site and bind with similar affinities (K-s = 2.2 and 1.7 mM for peptides I and II). While phosphoryl group transfer is the rate-limiting step in k(cat) for peptide I (5 s(-1)) at neutral pH, peptide II is converted to product by a kinetic mechanism in which phosphoryl group transfer (45 s(-1)) and product release (20 s(-1)) partially control this parameter. Significant solvent isotope effects on k(cat) (k(0)/k(n) approximate to 1.6) are observed for the phosphorylation of both peptides in 95% D2O. Proton inventories on k(cat) for peptide I are linear, indicating that the phosphoryl group transfer step is associated with a single proton transfer. Conversely, proton inventories on k(cat) for peptide II are ''bowed'' up, consistent with a ''virtual'' transition state in which phosphoryl group transfer and product release steps partially control this parameter. The lack of solvent isotope effects on k(cat)/K-peptide for both peptides can be explained by an equilibrium isotope effect on substrate binding that offsets the kinetic isotope effect for phosphoryl group transfer. In keeping with this proposal, the K-I for the inhibitor peptide, EAEIFEAIE, is 11 +/- 0.08 and 6.5 +/- 0.82 mM in 0 and 60% D2O, respectively. Fitting of the proton inventory plots using modified forms of the Gross-Butler equation provide intrinsic isotope effects of 1.7 and 3.6 for peptides I and II. The combined data are consistent with a mechanism involving either an acid-base catalyst or a conformational change preceding the release of products that is accompanied by the disruption of a single hydrogen donor-acceptor pair.