Mechanism of Adenlylate Kinases

本项目的目标是研究 腺苷酸酶的结构-功能关系 激酶（AK）通过遗传、生物化学 生物有机和生物物理技术。 (1)定点 诱变将用于鉴定参与的残基， 底物在基态和基态的结合 过渡态 现有的三个模型（来自 核磁共振，X射线和理论研究）将是至关重要的 测试. 目标是获得修订的或新的模型。 (2)点突变对AK构象的影响 将分两个阶段进行评估。 第一阶段是使用 差异蛋白质NMR，差异圆二色性， 盐酸胍诱导的变性、热稳定性和 用计算机绘图来评估可能的结构扰动 在突变体和潜在的结构作用， 残基 第二阶段是利用质子核磁共振评价 选择突变体中的构象扰动更多 数量上。 (3)上述模型将进一步测试， 通过“互补底物诱变”进行改进， 各种底物类似物。 目标是确认 酶残基和蛋白质之间的特异性相互作用 从衬底侧的衬底。

The objective of this project is to study the structure-function relationships of the enzyme adenylate kinase (AK) by a combination of genetic, biochemical, bioorganic, and biophysical techniques. (1) Site-directed mutagenesis will be used to identify the residues involved in the binding of substrates in the ground state and in the transition state. The three existing models (derived from NMR, X-ray, and theoretical studies) will be critically tested. The goal is to obtain a revised or a new model. (2) The effects of point mutation on the conformation of AK will be evaluated in two stages. The first stage is to use difference protein NMR, difference circular dichroism, guanidine-HCl-induced denaturation, thermal stability, and computer graphics to assess possible structural perturbations in the mutant and the potential structural roles of the residues. The second stage is to use proton NMR to evaluate conformational perturbations in selected mutants more quantitatively. (3) The above model will be further tested and refined by "complementary substrate mutagenesis" using various substrate analogues. The goal is to confirm the specific interactions between the enzymic residues and the substrates from the substrate side.