Stopped-Flow and 18O Exchange Studies of Partial Reactions in Sodium Pump Catalysis and Transport

9507018坠落活动或初级运输可以说是生物学中最重要的悬而未决的问题之一。在主动运输中，酶将存储分子中催化的能量释放与离子在膜上的移动相结合，从而与浓度梯度背道而驰。在哺乳动物中，储存分子通常是5‘-三磷酸-三磷酸腺苷。通过将一个磷酸基团从ATP转移到酶上，然后再转移到水中，从而加速能量智商的释放，因此这种类型的泵被称为P型离子动力ATPase。这个群的三个主要成员是根据他们所运输的离子命名的。作为消化过程的一部分，质子泵使胃酸化，其故障是溃疡的直接原因之一。钙泵通过从肌肉细胞的收缩装置周围的空间中移除钙离子来放松肌肉。钠泵通过将钠离子泵出细胞以换取钾离子来维持渗透平衡，几乎在所有细胞中都有这种泵。由初级转运体产生的钠梯度随后被次级转运体用来将糖等代谢物转移到细胞内。钠梯度也被用来刺激神经和肌肉细胞。钠泵是洋地黄药物的靶点，洋地黄是治疗充血性心力衰竭的主要药物。基本的研究问题是解释分解ATP释放的能量是如何用来泵送离子的，以及离子是如何物理转移的。主动转运领域的工作假说是，酶经历了一个构象变化的周期。目前尚不清楚这些三维形状变化的分子细节。例如，运输的离子是如何引发分子重排的，范围有多大？通过连接荧光报告基团可以直接观察到非磷酸化钠泵的构象变化。反应速度很快，因此必须通过在所谓的停流机中将标记的泵与几毫秒内的离子机械混合来启动。在之前的授权期中，研究表明，在构象发生变化之前，必须有两个钾离子与泵浦结合。在这个授权期内要检验的假设是，被传输的离子通过随机结合到相同和独立的位置来控制构象变化，而不是反合作地与离子井结合。第二个目标是通过测量分子上两点之间的距离变化来了解形状的变化有多大。这将通过同时将两个报告组连接到泵浦并测量它们之间的荧光能量转移来实现。第三个目标是测试有关磷酸化酶的中间构象数量及其在催化和运输中的作用的理论。这将通过合成三磷酸腺苷和含有稳定氧同位素的无机磷来完成。当磷酸酶形成并被水解时，稳定的同位素被水中的氧原子取代。用质谱仪可以测定产物中稳定同位素的分布和同位素交换速率。同位素分布取决于两个反应步骤发生的相对概率，因此，如果它随着钠浓度的变化而变化，则意味着钠改变了反应机理。第四个目标是利用同位素交换测量的更高的灵敏度和信息含量，以了解通过基因工程改变泵中的单个氨基酸如何影响分子的功能。蛋白质的构象变化是动态事件，即使是最灵敏的静态结构测定方法也不能完全理解。从基因工程蛋白质中可以了解到多少，取决于可用于评估氨基酸取代对功能影响的检测方法的敏感性。因此，这项拟议的研究还将推进对其他通过改变形状发挥功能的生物分子的研究。它将为博士后学生提供快速混合和稳定同位素交换技术的培训。钠泵通过将钠离子泵出细胞以换取钾离子来维持渗透平衡，几乎存在于所有哺乳动物细胞中。钠梯度随后被次级转运蛋白用来将代谢物转移到细胞内。基本的研究问题是解释存储能量分子裂解释放的能量如何用于泵浦离子，以及离子是如何物理转移的。工作假设是，这种酶经历了一个构象变化的周期。目前尚不清楚的是分子细节。一个要用快速反应方法检验的假设是，被传输的离子通过随机结合到相同和独立的位置来控制构象变化。第二个目标是通过荧光能量转移测量分子上两点之间的距离变化来了解形状的变化有多大。第三个目标是通过测量稳定的氧同位素交换来检验关于磷酸化酶中间构象数量的理论。第四个目标是利用同位素交换测量的更高的灵敏度和信息含量，以了解通过基因工程改变泵中的单个氨基酸如何影响分子的功能。***

9507018 Faller Active, or primary, transport is arguably one of the most important unsolved problems remaining in biology. In active transport, an enzyme couples the catalyzed release of energy from a storage molecule to the movement of ions across a membrane against a concentration gradient. In mammals the storage molecule is usually adenosine 5'-triphosphate ATP. Release of the energy iq accelerated by transferring a phosphate group from ATP to the enzyme before transferring it to water, so pumps of this type are called P-type ion motive ATPases. The three principle members of the group are named for the ion they transport. The proton pump acidifies the stomach as part of the digestive process, and its malfunction is one immediate cause of ulcers. The calcium pump relaxes muscles by removing calcium ions from the space around the contractile apparatus in muscle cells. The sodium pump maintains osmotic balance by pumping sodium ions out of cells in exchange for potassium ions and is found in nearly all cells. The sodium gradient generated by the primary transporter is then used by secondary transporters to move metabolites like sugar into cells. The sodium gradient is also used to excite nerve and muscle cells. The sodium pump is the target of the drug digitalis, a primary therapy in congestive heart failure. The basic research problem is to explain how the energy released by splitting ATP is used to pump ions and how the ions are physically translocated. The working hypothesis in the active transport field is that the enzyme undergoes a cycle of conformational changes. What is not known are the molecular details of these changes in three dimensional shape. For example, how do transported ions trigger the molecular rearrangement and how extensive is it? The conformational change il unphosphorylated sodium pump can be observed directly by attaching fluorescent reporter groups. The reaction is fast, so it must be initiated by mechanically mixing labeled pump with ions within a few millise conds in what is called a stopped-flow machine. In the preceding grant period it was shown that two potassium ions must bind to the pump before, the conformational change occurs. The hypothesis to be tested in this grant period is that the transported ions control the conformational change by randomly binding to identical and independent sites instead of anticooperatively to an ion well. A second objective is to learn how big the change in shape is by measuring how much the distance between two points on the molecule changes. This will be done by simultaneously attaching two reporter groups to the pump and measuring the transfer of fluorescence energy between them. A third objective is to test theories about the number of intermediate conformations of phosphorylated enzyme and their role in catalysis and transport. This will be done by synthesizing ATP and inorganic phosphate containing a stable isotope of oxygen. When the phosphoenzyme is formed and hydrolyzed, the stable isotope is replaced by oxygen atoms from water. The rate of isotope exchange and the distribution of stable isotope in the product can be measured by mass spectrometry. The isotopomer distribution depends on the relative probability of two reaction steps occurring, so if it changes with sodium concentration, for example, it means that sodium changes the mechanism of the reaction. A fourth objective is to exploit the greater sensitivity and information content of isotope exchange measurements to learn how changing individual amino acids in the pump by genetic engineering affects the molecule's function. Conformational changes in proteins are dynamic events that cannot be completely understood by even the most sensitive static methods for structural determination. How much can be learned from genetically engineering proteins depends on the sensitivity of assays available to assess the effect of amino acid substitutions on function. Therefore, the research that is proposed will also advance studies of other biological molecules that function by changing their shape. It will provide postdoctoral students with training in rapid mixing and stable isotope exchange techniques. %%% The sodium pump maintains osmotic balance by pumping sodium ions out of cells in exchange for potassium ions and is found in nearly all mammalian cells. The sodium gradient is then used by secondary transporters to move metabolites into cells. The basic research problem is to explain how the energy released by splitting an energy storage molecule is used to pump ions and how ions are physically translocated. The working hypothesis is that the enzyme undergoes a cycle of conformational changes. What is not known are the molecular details. One hypothesis to be tested by rapid reaction methods is that the transported ions control the conformational change by randomly binding to identical and independent sites. A second objective is to learn how big the change in shape is by fluorescence energy transfer measurements of the distance change between two points on the molecule. A third objective is to test theories about the number of intermediate conformations of phosphorylated enzyme by measuring stable oxygen isotope exchange. A fourth objective is to exploit the greater sensitivity and information content of isotope exchange measurements to learn how changing individual amino acids in the pump by genetic engineering affects the molecule's function. ***