Here we characterized this conformation using pure mouse MDR3 P-glycoprotein and natural MgATP and MgADP. Mutants E552A/E1197A, E552Q/E1197Q, E552D/ E1197D, and E552K/E1197K had low but real ATPase activity in the order Ala > Gln > Asp > Lys, emphasizing the requirement for Glu stereochemistry. Mutant E552A/ E1197A bound MgATP and MgADP (1 mol/mol) with K d 9.2 and 92 M, showed strong temperature sensitivity of MgATP binding and equal dissociation rates for MgATP and MgADP. With MgATP as the added ligand, 80% of bound nucleotide was in the form of ATP. None of these parameters was vanadate-sensitive. The other mutants showed lower stoichiometry of MgATP and MgADP binding, in the order Ala > Gln > Asp > Lys. We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized NBD dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly. Impairment of turnover is primarily due to inability to form the normal transition state rather than to slow ADP release. The Gln, Asp, and Lys mutants are less effective at stabilizing the occluded nucleotide, putative dimeric NBD, conformation. We envisage that in wild-type the occluded nucleotide conformation occurs transiently after MgATP binds to both NBDs with associated dimerization, and before progression to the transition state.P-glycoprotein (Pgp) 1 is a plasma membrane protein that confers multidrug resistance by virtue of its ability to exclude hydrophobic compounds from cells in an ATP-dependent fashion. Anticancer drugs and AIDS protease inhibitors are among the numerous compounds transported by Pgp, and there is the realization that a substantial number of future new drug candidates will similarly be transport substrates, so much current interest centers on strategies aimed at disabling or circumventing Pgp. Consisting of two transmembrane domains and two nucleotide binding domains (NBDs), Pgp displays the typical architecture of the ABC transporter family of membrane transporters. There is as yet no reported high resolution structure of Pgp, but structures of several homologous ABC transporters, and of isolated NBDs, have been published recently. For recent reviews of Pgp structure and function see Refs. 1-6.Our laboratory has focused on the mechanism of ATP hydrolysis and ATP hydrolysis-driven transport. Procedures for large scale preparation of pure human MDR1 (7) and mouse MDR3 2 protein (8) have recently facilitated such studies. Earlier work had established that Pgp showed drug-stimulated ATPase activity (9, 10) and that hydrolysis of ATP occurred in both NBDs (11). It was clear from studies with inhibitors N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (10, 12-14), mutations in the catalytic sites (15, 16), and vanadate-trapping experiments (17) that the two NBDs cooperated strongly and mandatorily for hydrolysis, and an alternating sites mechanism was proposed in 1995 (18). This mechanism envi...