title: Comparison of covalent with reversible inhibitor binding sites of the gastric H,K-ATPase by site-directed mutagenesis.
abstract: The gastric H,K-ATPase is covalently inhibited by substituted pyridyl-methylsulfinyl-benzimidazoles, such as omeprazole, that convert to thiophilic probes of luminally accessible cysteines in the acid space. The K(+) competitive 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. Mutations of Cys-822 and Cys-892 had insignificant effects on the K(i(app)), K(m(app)) 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 produced mixed kinetics of inhibition, still with higher affinity for the cation-free form of phosphoenzyme. Since the phenylmethoxy ring of the imidazo-pyridine 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-321 --> Ala) decrease the affinity for SCH28080, the TM1/2, TM3, and TM6 helices lie within approximately 16 A of each other based on the size of the active, extended conformation of SCH28080.
authors.affiliation: Department of Physiology, School of Medicine, UCLA and Veterans Affairs Greater Los Angeles Health Care System, Los Angeles, California 90073, USA.
authors.forename: N
authors.initials: N
authors.lastname: Lambrecht
authors.forename: K
authors.initials: K
authors.lastname: Munson
authors.forename: O
authors.initials: O
authors.lastname: Vagin
authors.forename: G
authors.initials: G
authors.lastname: Sachs
mesh_terms: D000897:Anti-Ulcer Agents / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D018613:Microscopy, Confocal; D008968:Molecular Conformation; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009853:Omeprazole / Q000494:pharmacology; D017433:Protein Structure, Secondary; D054328:Proton Pump Inhibitors; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology
chemical_list: D000897:Anti-Ulcer Agents; D004791:Enzyme Inhibitors; D007093:Imidazoles; D054328:Proton Pump Inhibitors; D000644:Quaternary Ammonium Compounds; C035235:Sch 28080; D017506:H(+)-K(+)-Exchanging ATPase; D009853:Omeprazole