Comparison of covalent with reversible inhibitor binding sites of the gastric H,K-ATPase by site-directed mutagenesis

The gastric H,K-ATPase is covalently inhibited by substituted pyridyl-methy lsulfinyl-benzimidazoles, such as omeprazole, that convert to thiophilic pr obes of luminally accessible cysteines in the acid space. The K+ competitiv e inhibitor, SCH28080, prevented inhibition of acid transport by omeprazole . In stably expressing HEK293 cells, the benzimidazole-reactive cysteines, Cys-321 (transmembrane helix (TM) 3), Cys-813 and Cys-822 (TM5/6), and Cys- 892 (TM7/8) were mutated to the amino acids found in the SCH28080-resistant Na,K-ATPase and kinetic parameters of H,K-ATPase activity analyzed. Mutati ons of Cys-822 and Cys-892 had insignificant effects:an the K-i(app), K-m(a pp) or V-max, but mutations of Cys-813 to threonine and Cys-321 to alanine decreased the affinity for SCH28080, Mutation of Cys-321 to alanine produce d mixed kinetics of inhibition, still with higher affinity for the cation-f ree form of phosphoenzyme. Since the phenylmethoxy ring of the imidazo-pyri dine inhibitors binds to TM1/2, as shown by earlier photoaffinity studies, and the mutations in TM6 (Cys-813 --> Thr) as well as the end of TM3 (Cys-3 21 --> Ala) decrease the affinity for SCH28080, the TM1/2, TM3, and TM6 hel ices lie within: similar to 16 Angstrom of each other based on the size of the active, extended conformation of SCH28080.