P-glycoprotein Catalytic Mechanism

Abstract

Kinetics of inhibition ofP-glycoprotein (Pgp) 1 is a prominent member of the ABCtransporter family of membrane proteins that uses the energy of ATP hydrolysis to exclude hydrophobic compounds from cells. In human, it was first recognized as a major potential obstacle to successful chemotherapy of cancer because of its expression in cancer cells and was later realized to function physiologically in such strategic locations as the blood-brain barrier, intestine, placenta, and elsewhere as an important agent in protection from environmental and dietary toxins. More recent appreciation of its role in impeding the action of anti-AIDS therapy and its potentially general role in reducing the efficacy of many hydrophobic drugs, new and old, have made it a target of intense investigation. Recent reviews of the biochemistry and pharmacology of Pgp may be found in Refs. 1-6.Our research has focused on the catalytic mechanism by which ATP is hydrolyzed and the energy transduced into drug transport. In 1995, we presented schemes for the hydrolysis of ATP at a single catalytic site (7), also for the interaction of the two ATP-binding sites in catalysis and transduction of the energy of hydrolysis to the drug binding site(s) situated in the membrane bilayer (8). Both ATP-binding sites were shown to have catalytic capability. Release of product P i was thought to occur before the release of ADP, which was rate-limiting. Interaction of the two ATP-binding sites was concluded to be an integral and a necessary facet of catalysis with hydrolysis of ATP occurring sequentially and alternately at each site. Formation and collapse of the transition state of catalysis were postulated as critical events in driving the changes in drugbinding site affinity and orientation (inward versus outward facing) necessary for binding and extrusion of drug with a stoichiometry of one ATP hydrolyzed per change in drug-binding site affinity and orientation. Later studies of Pgp and also of ABC transporter homologs such as bacterial LmrA and the maltose transporter have supported and extended the earlier schemes (9 -11). New ideas incorporated into more recent schemes include the concept of reciprocating pairs of drugbinding sites (10) and of the requirement for a second ATP hydrolysis event to "reset" the drug-binding site for drug extrusion (9). Another concept, recently introduced, envisages that the ATP-binding event provides the primary driving force for transport (12). Electron microscopy studies provide evidence for conformational changes in the membrane domains observed upon nucleotide binding (12); however, biochemical evidence has been provided showing that the drug-binding site also changes conformation in response to ATP hydrolysis per se (13,14).Recent work (15) demonstrated that, at concentrations of MgATP and MgADP present in the cytoplasm of mammalian cells, both nucleotide-binding domains (NBD) in "resting" Pgp would be expected to bind MgATP (15). Our original scheme for ATP-driven drug transport (8) envisaged that upon attainment of...

Combined Mutation of Catalytic Glutamate Residues in the Two Nucleotide Binding Domains of P-glycoprotein Generates a Conformation That Binds ATP and ADP Tightly

“…As noted in Ref. 22 and confirmed here, recovery of Pgp was routinely 70 -90% as judged by ATPase and/or protein assays. To reduce the amount of experimental manipulation to manageable proportions we routinely assumed a protein recovery of 80%.…”

“…It was suggested that the closed conformation was required after initial ATP binding to attain the transition state. Later work on formation of the transition state analog Pgp⅐MgADP⅐V i supported this proposal (22) as did cross-linking studies showing proximity (7) and likely interdigitation (23) of the Pgp NBDs. Studies with other ABC transporters have given structural backing to the concept.…”

“…Centrifuge column elution of Pgp was exactly as in Ref. 22. Briefly, elution was at 4°C, using 1-ml Sephadex G-50 columns equilibrated in 50 mM Tris-Cl, pH 7.5, and 0.001% N-dodecyl-␤-D-maltoside.…”

“…Previous experiments had indicated that the longer time is needed to attain saturation with ADP in the presence of V i (22).…”

Natural Phosphatidylcholine Is Actively Translocated across the Plasma Membrane to the Surface of Mammalian Cells

“…Inhibitors of these transporters reduced C 6 -NBD-PC outward translocation by an endogenous translocase in erythrocytes (23,46) and fibroblasts. 2 The inhibition of natural PC cell surface translocation in erythrocytes and fibroblasts by glibenclamide and vanadate, both general inhibitors of ABC transporters that affect ATP at the nucleotide-binding folds (65)(66)(67), could indicate that active translocation of endogenous PC in non-hepatic cells is also mediated by ABC transporters. In contrast to C 6 -NBD-PC, natural PC translocation in human erythrocytes was insensitive toward the more specific ABC transporter inhibitors PSC833 and MK571, suggesting that ABCB1 and ABCC1 are involved in C 6 -NBD-PC but not in natural PC translocation.…”