Our goal is to learn how the gastric proton pump works in molecular detail. It has been demonstrated that the adenosine 5'-triphosphatase (ATPase) isolated in the microsomal fraction from gastric mucosae is necessary and sufficient for coupling the energy released during ATP hydrolysis to the transport of protons in exchange for K+ ions. However, neither the molecular biology, nor the underlying chemistry of the reaction is understood. This proposal focuses on the number of nucleotide sites and their function, the identity of the subunits and quaternary structure of the enzyme, the role of Mg2+ in catalysis and transport, and the involvement of a protein conformational change in the energetics of phosphoenzyme formation and physical transport of ions. The strategy proposed to learn the number of nucleotide sites and their function is to build up a library of site-selective, covalent modifications of the H,K-ATPase. The effect of each reagent on the reactions catalyzed by the enzyme will be studied and the site of covalent modification pinpointed by sequencing the labeled peptide. This approach will also help to resolve the issue of subunit heterogeneity. Evidence for interaction between subunits will be obtained by studying their thermal denaturation. Mg2+ is the only cofactor required for formation of an acylphosphate enzyme intermediate. Therefore it must play a key role in the catalytic mechanism. The number of Mg2+ sites will be determined, and which ones are involved in catalysis will be established. The distance from the nucleotide site(s) to the K+ activation and transport sites will be measured. The thermodynamics of ligand-binding will be studied to learn whether a protein conformational change is triggered that could explain catalysis and transport. This work has implications for the treatment of ulcer disease. Substituted benzimidazoles have been found to inhibit gastric acid secretion in vivo and several are currently undergoing clinical trials for the treatment of peptic ulcers. The target of these drugs is the H,K-ATPase, but how they work is unknown. More detailed knowledge of the mechanism of catalysis and transport is needed both to understand how this new class of existing drugs inhibits acid secretion, and to design more effective inhibitors of the gastric proton pump in the future.
