P-glycoprotein catalytic mechanism: studies of the ADP-vanadate inhibited state.
Kinetics of inhibition of ATPase activity of pure mouse Mdr3 P-glycoprotein upon incubation with MgADP and vanadate were studied along with the trapping of [14C]ADP in presence of vanadate. The presence of verapamil strongly magnified both effects. Inhibition of ATPase was also increased by several other drugs known to bind to drug-binding sites. Inhibition by ADP-vanadate was slow and depended cooperatively on nucleotide binding. Stoichiometry of [14C]ADP trapping by vanadate was 1 mol/mol P-glycoprotein at full inhibition. Catalytic site mutants prevented [14C]ADP trapping, whereas interdomain signal communication mutants reduced it in approximate correlation with their effects upon drug stimulation of ATPase. In explanation of the results, we propose that a "closed conformation" involving dimerization and interdigitation of the two nucleotide-binding domains is necessary to allow inhibition by ADP-vanadate. The results suggest that such a conformation occurs naturally during ATP hydrolysis. It is proposed that in order for the catalytic transition state to form, the two nucleotide-binding domains dimerize to form an integrated single entity containing two bound ATP with just one of the two ATP being hydrolyzed per dimerization event.
Urbatsch
Ina L
IL
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
Tyndall
Grace A
GA
Tombline
Gregory
G
Senior
Alan E
AE
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism* / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D019281:Dimerization; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology*; D014700:Verapamil / Q000494:pharmacology
D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D011994:Recombinant Proteins; D014638:Vanadates; D000244:Adenosine Diphosphate; C095122:multidrug resistance protein 3; D014700:Verapamil; D000251:Adenosine Triphosphatases