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

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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. ***