Evidence for a requirement for ATP hydrolysis at two distinct steps duringa single turnover of the catalytic cycle of human P-glycoprotein

P-glycoprotein (Pgp) is an ATP-dependent hydrophobic natural product antica ncer drug efflux pump whose overexpression confers multidrug resistance to tumor cells. The work reported here deals with the elucidation of the energ y requirement for substrate interaction with Pgp during the catalytic cycle . We show that the K-d (412 nM) of the substrate analogue [I-125]iodoarylaz idoprazoin for Pgp is not altered by the presence of the nonhydrolyzable nu cleotide 5'-adenylylimididiphosphate and vanadate (K-d = 403 nM), Though bi nding of nucleotide per se does not affect interactions with the substrate, ATP hydrolysis results in a dramatic conformational change where the affin ity of [I-125]iodoarylazidoprazoin for Pgp trapped in transition-state conf ormation (Pgp . ADP . vanadate) is reduced >30-fold. To transform Pgp from this intermediate state of low affinity for substrate to the next catalytic cycle, i.e., a conformation that binds substrate with high affinity, requi res conditions that permit ATP hydrolysis. Additionally, there is an invers e correlation (R-2 = 0.96) between 8AzidoADP (or ADP) release and the recov ery of substrate binding. These results suggest that the release of nucleot ide is necessary for reactivation but not sufficient. The hydrolysis of add itional molecule(s) of ATP (or 8AzidoATP) is obligatory for the catalytic c ycle to advance to completion. These data are consistent with the observed stoichiometry of two ATP molecules hydrolyzed for the transport of every su bstrate molecule. Our data demonstrate two distinct roles for ATP hydrolysi s in a single turnover of the catalytic cycle of Pgp, one in the transport of substrate and the other in effecting conformational changes to reset the pump for the next catalytic cycle.