Functionally similar vanadate-induced 8-azidoadenosine 5 '-[alpha-P-32]diphosphate-trapped transition state intermediates of human P-glycoprotein aregenerated in the absence and presence of ATP hydrolysis

P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump whose overexpress ion confers multidrug resistance to cancer cells. Pgp exhibits a robust dru g substrate-stimulable ATPase activity, and vanadate (Vi) blocks this activ ity effectively by trapping Pgp nucleotide in a noncovalent stable transiti on state conformation. In this study we compare Vi-induced [alpha-P-32]8-az ido-ADP trapping into Pgp in the presence of [alpha-P-32]8-azido-ATP (with ATP hydrolysis) or [alpha-P-32]8-azido-ADP (without ATP hydrolysis), Vi mim ics Pi to trap the nucleotide tenaciously in the Pgp . [alpha-P-32]8-azido- ADP.Vi conformation in either condition. Thus, by using [alpha-P-32]8-azido -ADP we show that the Vi-induced transition state of Pgp can be generated e ven in the absence of ATP hydrolysis. Furthermore, half-maximal trapping of nucleotide into Pgp in the]presence of Vi occurs at similar concentrations of [(alpha-P-32]8-azido-ATP or [alpha-P-32]8-azido-ADP. The trapped [(alph a-P-32]8-azido-ADP is almost equally distributed between the N- and the C-t erminal ATP sites of Pgp in both conditions. Additionally, point mutations in the Walker B domain of either the N- (D555N) or C (D1200N)-terminal ATP sites that arrest ATP hydrolysis and Vi-induced trapping also show abrogati on of [alpha-P-32]8-azido-ADP trapping into Pgp in the absence of hydrolysi s, These data suggest that both ATP sites are dependent on each other for f unction and that each site exhibits similar affinity for 8-azido-ATP (ATP) or 8-azido-ADP (ADP), Similarly, Pgp in the transition state conformation g enerated with either ADP or ATP exhibits drastically reduced affinity for t he binding of analogues of drug substrate ([I-125]iodoarylazidoprazosin) as well as nucleotide (2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphospha te). Analyses of Arrhenius plots show that trapping of Pgp with [alpha-P-32 ]8-azido-ADP tin the, absence of hydrolysis) displays an similar to2.5-fold higher energy of activation (152 kJ/mol) compared with that observed when the transition state intermediate is generated through hydrolysis of [alpha -P-32]8-azido-ATP (62 kJ/mol), In aggregate, these results demonstrate that the Pgp [alpha-P-32]8-azido-ADP (or ADP) Vi transition state complexes gen erated either in the absence of or accompanying [alpha-P-32]8-azido-ATP hyd rolysis are functionally indistinguishable.