The adenosine 5'-triphosphatase (ATPase) isolated in the microsomal fraction from gastric mucosas is necessary and sufficient for acid secretion into the stomach. Hypersecretion is the immediate cause of ulcers. A new generation of drugs for the pharmacological management of the disease reduces acid secretion by inhibiting the gastric ATPase. Detailed knowledge of the enzyme's structure and mechanism were needed to understand how the first drugs in this class inactivated the enzyme and even more detailed information will be required to develop more effective agents with fewer side effects. The long term goal of this research is to explain how the gastric ATPase catalyzes ATP hydrolysis and couples the energy released to proton transport across the gastric epithelium. That means learning the structure of the enzyme and relating it to function. This proposal focuses on the role of protons in the catalytic mechanism, a peptide that may be a functional subunit of the enzyme, identification of functional amino acids, and conformational changes in the protein that may be the key to energy coupling and the physical transport of ions. A change in the distribution of oxygen isotopes in inorganic phosphate when it reacts with the enzyme in solutions of different acidity indicates that a weakly basic group on the enzyme functions in catalysis. Which step in the catalytic mechanism is affected by protonating this group will be assessed by measuring the rates of the individual steps in the reaction. Chemical modification of a peptide that has been isolated from gastric microsomes inactivates the ATPase. The peptide will be sequenced and the hypothesis that it forms a functional complex with the enzyme will be tested. Amino acids in the enzyme's active site will be identified by chemical modification with a substrate analogue, isolation of the labeled peptide, and microsequence analysis of its structure. Three conformational changes in the gastric enzyme have been observed with fluorescent reporter groups. The rates of these changes will be measured and compared with the rates of steps in the catalytic cycle to learn which changes in structure are functional.
