FUNCTIONAL ANALYSIS OF A LEAD/CADMIUM/ZINC ATPASE

铅/镉/锌ATP酶的功能分析

• 批准号: 6327506
• 负责人: BHARATI MITRA
• 金额: $ 18.83万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6327506
• 关键词: X ray crystallography; adenosinetriphosphatase; bacterial proteins; cadmium; chelating agents; chemical kinetics; crystallization; enzyme reconstitution; fluorescence resonance energy transfer; heavy metals; intermolecular interaction; ion transport; lead; model design /development; molecular shape; molecular site; molecular size; phosphorylation; physical model; protein structure function; site directed mutagenesis; structural biology; zinc

DESCRIPTION: (Applicant's Description) The superfamily of P-type ATPases catalyzes the energy-dependent transport of charged substrates across membranes. These ubiquitous pumps transport different cations and carry out a wide variety of functions, for example, they generate the transmembrane ion gradients necessary for nutrient uptake, signal transduction, and maintenance of suitable pH and ion concentrations as well as maintain the asymmetric distribution of phospholipids across plasma membranes of animal cells. Soft-metal P-type ATPases maintain homeostasis of the essential metals, Cu(I), Zn (II) and Co(II) and also mediate resistance to Ag(I), Pb(II), Cd(II), and other highly toxic metal cations. The human Cu(I)-transporting ATPases associated with Menkes and Wilson diseases, are examples of monovalent soft-metal ATPases whereas ZntA from Escherichia coli, the focus of this application, is a divalent soft-metal ATPase. The objective of this proposal is to use ZntA as a prototype to study the mechanism by which soft-metal ATPases recognize and transport specific metal ions. ZntA is ideal for this study because it has been cloned, overexpressed, solubilized and purified. An ATP-dependent transport assay has been developed for ZntA. A soft-metal dependent ATP hydrolysis assay has been optimized; ZntA is the first soft-metal ATPase for which this activity has been demonstrated. The specific aims include biochemical characterization of ZntA and analysis of the contributions of conserved residues and different domains, including the cysteine-rich amino-terminal domain of soft-metal ATPases, towards metal recognition and transport. The interaction between the metal-binding and transport domains with the ATP hydrolysis domain will be examined. The coordination and geometry adopted by different metal ions will be studied. Efforts to address these specific aims will include transport, ATP hydrolysis and phosphoenzyme formation assays, site-specific mutagenesis, and fluorescence EXAFS and x-ray structural studies.

描述：（申请人的描述）P型ATPASE的超家族 催化带电底物跨的能量依赖性运输 膜。这些无处不在的泵运输不同的阳离子并进行 例如，它们生成跨膜离子的多种功能 营养吸收，信号转导和维护所需的梯度 合适的pH和离子浓度以及保持不对称的 跨动物细胞质膜的磷脂分布。 软金属P型ATPases维持基本金属的稳态，Cu（i）， Zn（II）和CO（II），还介导对Ag（I），Pb（II），CD（II）和 其他剧毒金属阳离子。人类铜（i）传输ATPases 与Menkes和Wilson疾病相关的是单价的例子 软金属ATPase，而Znta来自大肠杆菌，重点 应用是二价软金属ATPase。 该提案的目的是将ZNTA用作原型来研究 软金属ATPases识别和运输特定金属的机制 离子。 ZNTA是本研究的理想选择，因为它已被克隆，过表达， 溶解和纯化。已经开发了依赖ATP的运输测定法 对于Znta。 软金属依赖性ATP水解测定法已优化； Znta是 已经证明了该活动的第一个软金属ATPase。 具体目的包括ZNTA的生化表征和分析 保守残留物和不同领域的贡献，包括 软金属ATPases的富含半胱氨酸的氨基末端结构域，朝金属 认可和运输。金属结合与 将检查具有ATP水解结构域的运输域。这 将研究不同金属离子采用的协调和几何形状。 解决这些特定目标的努力将包括运输，ATP水解 和磷酸酶的形成测定，位点特异性诱变和 荧光EXAF和X射线结构研究。