A variety of life processes depend on the selective transport of ions across cell membranes against a concentration gradient. Despite the importance of this phenomenon, the most fundamental questions about the molecular mechanism of active transport remain unanswered. The objective of the proposed research is to learn how ion transport is coupled to catalysis of energy release from the biological storage molecule (ATP) by the class of enzyme that forms a phosphoenzyme intermediate (E1E2-type). The current theory is that phosphorylation by ATP and by inorganic phosphate (P1) are catalyzed by different conformations of the enzyme. This idea will be tested by studying oxygen-18 exchange, which is catalyzed when the enzyme is phosphorylated. The rate of appearance and disappearance of inorganic phosphate containing from zero to four oxygen-18 atoms can be measured by mass spectrometry or nuclear magnetic resonance. The pattern of isotope exchange depends upon the mehcanism of the reaction. It has been found to change dramatically when the concentration of one of the ions transported by the proton, potassium-ATPase (H+) is increased. Therefore, the relationship between conformational state and mechanism can be sensitively probed by measuring the isotope exchange pattern. The enzyme that catalyzes sodium, potassium transport will also be studied, because there is a wealth of information on how Na+ and K+ affect the conformation of the enzyme. The primary model to be used in these studies, the gastric proton, potassium-ATPase, is important physiologically as well as theoretically.
