Evidence for the vectorial nature of drug (substrate)-stimulated ATP hydrolysis by human P-glycoprotein

P-glycoprotein (Pgp), the ATP-binding cassette multidrug transporter, exhib its a drug (substrate)-stimulatable ATPase activity, and vanadate (Vi) inhi bits this activity by stably trapping the nucleoside diphosphate in the Pgp -ADP-Vi conformation. We recently demonstrated that Vi-induced 8-azido-[alp ha-(32)p]ADP trapping into Pgp in the absence of substrate occurs both in t he presence of 8-azido-[alpha-P-32]ATP (following 8-azido-ATP hydrolysis) o r 8-azido-[alpha-P-32]ADP (without hydrolysis) and, the transition state in termediates generated under either condition are functionally indistinguish able. In this study, we compare the effect of substrates on Vi-induced 8-az ido.[alpha-P-32]ADP trapping into Pgp under both non-hydrolysis and hydroly sis conditions. We demonstrate that whereas substrates stimulate the Vi-ind uced trapping of 8-azido-[alpha-P-32]ADP under hydrolysis conditions, they strongly inhibit Vi-induced trapping under non-hydrolysis conditions. This inhibition is concentration-dependent, follows first order kinetics, and is effected by drastically decreasing the affinity of nucleoside diphosphate for Pgp during trapping. However, substrates do not affect the binding of n ucleoside diphosphate in the absence of Vi, indicating that the substrate-i nduced conformation exerts its effect at a step distinct from nucleoside di phosphate-binding. Our results demonstrate that during the catalytic cycle of Pgp, although the transition state, Pgp.ADP.P-i (Vi), can be generated b oth via the hydrolysis of ATP or by directly providing ADP to the system, i n the presence of substrate the reaction is driven in the forward direction , i.e. hydrolysis of ATP. These data suggest that substrate-stimulated ATP hydrolysis by Pgp is a vectorial process.