STRUCTURE/FUNCTION RELATIONSHIPS OF GUANYLATE KINASE

鸟苷酸激酶（GK）在cGMP循环中起着重要作用， 参与鸟嘌呤核苷酸介导的信号转导途径。 该项目的长期目标是建立定量结构- 酵母GK的功能关系，将占其催化 机制和核苷酸特异性，使用动力学和 热力学方法、定点突变和生物物理方法 方法. 项目的具体目标包括：（1）确定 GK催化反应的动力学途径和能量学。 状态和瞬态动力学和热力学测量。 目标 是获得一个完整的自由能分布，这将是基础， 剖析单个氨基酸残基对 催化和底物特异性。 (2)为了评估 催化活性位点残基和核苷酸特异性位点- 定向诱变 这些残基对每一步的贡献 将通过评估突变的影响来量化催化作用 具体目标1中所述的反应曲线。 此外，如何 与底物相互作用的氨基酸残基将通过 检查底物类似物的动力学和/或立体化学。 (3)评估突变对结构和稳定性的影响 通过生物物理方法。 目标不仅是确定 氨基酸残基的结构和构象稳定性，而且 为定量分析提供必要的结构信息， 具体目标2中获得的结果的解释。 (4)到 通过以下方式鉴定与结合的ATP非常接近的氨基酸残基： 通过NMR将核苷酸对接到酵母GK的晶体结构中， 计算机图形与距离的限制，从核磁共振数据。 （五） 设计新的底物特异性。 最初的目标是重新设计 GMP位点，使酶催化磷酸化 从ATP特异性转移至IMP、XMP或AMP。 ATP网站将 在项目的后期阶段也要进行修改， GTP、ITP或XTP。

Guanylate kinase (GK) plays an essential role in the cGMP cycle and may be involved in guanine nucleotide-mediated signal transduction pathways. The long-term goal of the project is to establish quantitative structure- function relationships for yeast GK that will account for its catalytic mechanism and nucleotide specificity, using a combination of kinetic and thermodynamic methods, site-directed mutagenesis, and biophysical methods. The specific aims of the project include: (1) To determine the kinetic pathway and energetics of the GK-catalyzed reaction by steady- state and transient kinetics and thermodynamic measurements. The goal is to obtain a complete free energy profile which will be the basis for dissecting the contributions of individual amino acid residues to catalysis and substrate specificity. (2) To evaluate the roles of the active site residues in catalysis and nucleotide specificity by site- directed mutagenesis. The contributions of these residues to each step of catalysis will be quantitated by evaluating the effects of mutations on the reaction profile as described in Specific Aim 1. Furthermore, how the amino acid residues interact with the substrates will be probed by examining the kinetics and/or stereochemistry of substrate analogues. (3) To assess the effects of mutations on the structure and stability by biophysical methods. The goal is not only to define the roles of the amino acid residues in structure and conformational stability but also to provide the essential structural information for quantitative interpretation of the results obtained in Specific Aim 2. (4) To identify the amino acid residues in close proximity to the bound ATP by NMR and dock the nucleotide into the crystal structure of yeast GK by computer graphics with the distance constraints from the NMR data. (5) To engineer new substrate specificity. The initial goal is to redesign the GMP site by mutagenesis so that the enzyme catalyzes phosphoryl transfer from ATP specifically to IMP, XMP, or AMP. The ATP site will also be modified at the late stage of the project so that it is specific for GTP, ITP, or XTP.