NMR STUDIES OF PHOSPHORYL TRANSFER ENZYMES

磷酸基转移酶的核磁共振研究

• 批准号: 3467747
• 负责人: ENGIN H SERPERSU
• 金额: $ 8.21万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3467747
• 关键词: adenosine triphosphate; chemical association; chemical binding; chemical group; chemical kinetics; divalent cations; electron spin resonance spectroscopy; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; isomorphous substitution; ligands; molecular dynamics; nuclear magnetic resonance spectroscopy; nucleotide analog; phosphoglycerate; phosphoglycerate kinase; plasmids; protein structure function; radionuclides; site directed mutagenesis; stoichiometry; yeasts

The long range objective of the proposed study is to learn detailed mechanism by which enzyme-catalyzed nucleophilic substitution occurs at phosphorus and to clearly define the structural requirements for the formation of a functional enzyme-substrate(s) complex by studies with phosphoglycerate kinase (PGK). PGK, likely catalyzes the reaction via a hinge-bending motion of two domains, which results in the closure of an active site cleft, as suggested by x-ray studies. However, mechanism of the enzyme is not known at a chemical and structural level. This proposal will examine the mechanism of catalysis. The active site structure and its relationship to catalysis with PGK by studying the conformations and the arrangements of he substrates and substrate analogs, the coordination number and the position of the activator metal ion with respect to substrates at the active site of PGK. The identities and the roles of the amino acid residues near the substrates will be determined and their qualitative and quantitative contributions to catalysis will be studied. These goals will be achieved by focusing on 5 specific aims: 1) The arrangement and conformation of substrates at the active site will be determined by measuring the distances to substrate nuclei from a paramagnetic reference point, such as an activator metal ion Mn2+ or Co2+ or a paramagnetic substrate analog Cr(III) ATP. 2) Intramolecular nuclear Overhauser effect studies (NOE) will be performed to define the sugar pucker and glycosidic torsional angle (chi) of the nucleotide at the active site. Combined use of the data in aims 1 and 2 will provide more precise description of the conformation of ATP (or AMPP-CH2-P) in the different complexes of enzyme, metal, ATP and 3-PGA. 3) Detailed knowledge of structure of metal AMPP-CH2-P-3-PGA will be completed by determining coordination number of the metal ion by NMR and Electron Spin Echo Modulation Studies in the various complexes of enzyme, metal, AMPP-CH2-P and 3-PGA. 4) The identities of amino acid residues near substrate protons will be determined by intermolecular NOE studies in the diamagnetic complexes of enzyme, Mg2+, AMPP-CH2-P and 3-PGA (see the preliminary results). The distances from paramagnetic metal ion to these amino acids will be determined in separate experiments by using totally deuterated enzyme with only the designated amino acid in protonated form to yield visible NMR spectrum. 5) The roles of catalytically important amino acids in catalysis (determined in 4) will be tested by site specific mutations at these positions. These mutants will also be studied in detail by using the same nuclear magnetic resonance methods mentioned above. The proposed studies are both necessary and fundamental to our understanding of the catalysis by phosphoglycerate kinase and the results of these studies may provide useful information for the studies with the other kinases, which catalyze reactions by a similar hinge-bending action. This approach may be useful to elucidate general principles of enzyme chemistry, and to develop and critically compare various spectroscopic approaches to enzyme structure and mechanism.

拟议的研究的远距离目标是学习详细 酶催化的亲核取代的机制发生在 磷并清楚地定义了 通过研究的研究 磷酸甘油酸激酶（PGK）。 PGK，可能通过A催化反应 两个域的铰链弯曲运动，导致闭合 正如X射线研究所建议的那样，主动部位裂口。 但是，机制 该酶在化学和结构水平上尚不清楚。 这个建议 将检查催化的机制。 主动地点结构及其 通过研究构象和催化与PGK的关系 HE底物和底物类似物的排列，协调 相对于激活剂金属离子的位置 PGK活跃部位的底物。 身份和角色 将确定底物附近的氨基酸残基，并 将研究对催化的定性和定量贡献。 这些目标将通过关注5个特定目标来实现：1） 在活动地点的基板的布置和构象将是 通过测量从A的底物核的距离来确定 顺磁参考点，例如激活剂金属离子MN2+或CO2+ 或顺磁性底物类似物CR（III）ATP。 2）分子内核 将进行过度肝效应研究（NOE）以定义糖 活性核苷酸的冰球和糖苷扭转角（CHI） 地点。 目标1和2中数据的合并使用将提供更精确的 在不同的ATP（或AMPP-CH2-P）构象的描述 酶，金属，ATP和3-PGA的复合物。 3）详细了解 金属AMPP-CH2-P-3-PGA的结构将通过确定完成 NMR和电子自旋回波的金属离子的配位数 酶，金属，AMPP-CH2-P的各种复合物的调节研究 和3-PGA。 4）底物质子附近的氨基酸残基的身份 将由二磁性的分子间NOE研究确定 酶，MG2+，AMPP-CH2-P和3-PGA的复合物（请参阅初步 结果）。 从顺磁金属离子到这些氨基酸的距离 将在单独的实验中使用完全剥离来确定 仅用质子化形式的指定氨基酸的酶以产生 可见的NMR光谱。 5）催化重要的氨基酸的作用 在催化中（在4中确定）将通过位点特定突变测试 这些职位。 这些突变体也将通过使用 上面提到的相同的核磁共振方法。 拟议的研究既是我们的必要和基础 了解磷酸甘油酸激酶的催化和结果 这些研究可能为研究提供了有用的信息 其他激酶，通过类似的铰链弯曲作用催化反应。 这种方法可能有助于阐明酶的一般原理 化学，并开发和批判性比较各种光谱 酶结构和机制的方法。