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J Biol Chem, Vol. 275, Issue 6, 4041-4048, February 11, 2000

Comparison of Covalent with Reversible Inhibitor Binding Sites of the Gastric H,K-ATPase by Site-directed Mutagenesis*

Nils Lambrecht, Keith Munson, Olga Vagin, and George SachsDagger

From the Department of Physiology, School of Medicine, UCLA and Veterans Affairs Greater Los Angeles Health Care System, Los Angeles, California 90073

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 Ki(app), Km(app) or Vmax, 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 right-arrow Thr) as well as the end of TM3 (Cys-321 right-arrow Ala) decrease the affinity for SCH28080, the TM1/2, TM3, and TM6 helices lie within ~ 16 Å of each other based on the size of the active, extended conformation of SCH28080.

* This work was supported in part by National Institutes of Health Grants DK40615, 41301, and 17294 and by a United States Department of Veterans Affairs Senior Medical Investigator grant.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom all correspondence should be addressed: Bldg. 113, Rm. 326, Veterans Affairs Greater Los Angeles Health Care System, Los Angeles, CA 90073. Fax: 310-312-9478.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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