Correlation between steady-state ATP hydrolysis and vanadate-induced ADP trapping in human P-glycoprotein - Evidence for ADP release as the rate-limiting step in the catalytic cycle and its modulation by substrates

P-glycoprotein (Pgp) is a transmembrane protein conferring multidrug resist ance to cells by extruding a variety of amphipathic cytotoxic agents using energy from ATP hydrolysis. The objective of this study was to understand h ow substrates affect the catalytic cycle of ATP hydrolysis by Pgp, The ATPa se activity of purified and reconstituted recombinant human Pgp was measure d using a continuous cycling assay. Pgp hydrolyzes ATP in the absence of dr ug at a basal rate of 0.5 mu mol.min.mg(-1) with a K-m for ATP of 0.33 mM. This basal rate can be either increased or decreased depending on the Pgp s ubstrate used, without an effect on the K-m for ATP or 8-azidoATP and K-i f or ADP, suggesting that substrates do not affect nucleotide binding to Pgp, Although inhibitors of Pgp activity, cyclosporin A, its analog PSC833, and rapamycin decrease the rate of ATP hydrolysis with respect to the basal ra te, they do not completely inhibit the activity. Therefore, these drugs can be classified as substrates. Vanadate (Vi)-induced trapping of [alpha-P-32 ]8-azidoADP was used to probe the effect of substrates on the transition st ate of the ATP hydrolysis reaction. The K-m for [alpha-P-32]8-azidoATP (20 muM) is decreased in the presence of Vi; however, it is not changed by drug s such as verapamil or cyclosporin A. Strikingly, the extent of Vi-induced [alpha-P-32]8-azidoADP trapping correlates directly with the fold stimulati on of ATPase activity at steady state, Furthermore, P-i exhibits very low a ffinity for Pgp (K(i)similar to 30 mM for Vi-induced 8-azidoADP trapping). In aggregate, these data demonstrate that the release of Vi trapped [alpha- P-32]8-azidoADP from Pgp is the rate-limiting step in the steady-state reac tion. We suggest that substrates modulate the rate of ATPase activity of Pg p by controlling the rate of dissociation of ADP following ATP hydrolysis a nd that ADP release is the rate-limiting step in the normal catalytic cycle of Pgp.