Residues in P-glycoprotein catalytic sites that react with the inhibitor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.
7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is a specific covalent inhibitor of P-glycoprotein ATPase activity (M. K. Al-Shawi, and A. E. Senior, 1993, J. Biol. Chem. 268, 4197-4206). Complete inhibition occurs at a reaction stoichiometry of 1 mol NBD/mol P-glycoprotein, and the reagent has proved valuable in understanding catalytic mechanisms, particularly in relation to catalytic site cooperativity (A. E. Senior, and S. Bhagat, 1998, Biochemistry 37, 831-836). The actual location of reaction in the amino acid sequence has not yet been determined. Using a combined mutagenesis and biochemical approach we establish here that the initial reaction of NBD-Cl is with Cys within the Walker A consensus sequence of the N- or C-terminal nucleotide site (Cys-431 or Cys-1074 of human P-glycoprotein). Reaction with either Cys yields full inhibition. It was further found that inhibition consists of dithiothreitol (DTT)-reversible and DTT-irreversible components. The former predominates at low pH and the latter at higher pH. This demonstrates that, at higher pH, intramolecular transfer of NBD from Cys to Lys occurs, probably to the proximate Walker A Lys (Lys-433 or Lys-1076 of human P-glycoprotein). After transfer of NBD to Lys, P-glycoprotein ATPase remains fully inhibited.
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
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Senior
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Gros
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Urbatsch
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology*; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000235:genetics; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000818:Animals; D016466:CHO Cells; D002250:Carbon Radioisotopes; D002384:Catalysis / Q000187:drug effects; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D004229:Dithiothreitol / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D002250:Carbon Radioisotopes; C095122:multidrug resistance protein 3; D000251:Adenosine Triphosphatases; D009327:4-Chloro-7-nitrobenzofurazan; D008239:Lysine; D004229:Dithiothreitol