Exploiting enzymatic transition states of the ATPase reaction to elucidate the mechanism of transport by P‐glycoprotein (ABCB1)

The ATP‐binding cassette (ABC) family of transport proteins is one of the largest families of proteins in living organisms. Understanding how these proteins work is extremely important because of their central role in biology and the fact that many members of this group are implicated in human diseases. The transport cycle of P‐glycoprotein (Pgp), the archetypical ABC transporter, has received considerable attention, but it has not been possible to reconcile the data from various laboratories into a single model that explains the mechanism of transport. One of the key unresolved issues is whether it is the nucleotide binding alone or nucleotide hydrolysis that provides the “power‐stroke” for the pump. We have used the Walker B double mutant of Pgp (E556Q/E1201Q) or orthovanadate to capture the E·S and E·P transition states of Pgp respectively and characterized these transition states in terms of their thermodynamic, kinetic and physical properties. Such a defined framework allowed us to be precise in elucidating the steps in the ATPase reaction that convert the high affinity transport substrate site to a low affinity site. We demonstrate here that these conformational changes occur during the formation of the E·S transition state. The E·S state represents a reaction intermediate in the Pgp‐mediated ATPase reaction, hence ATP hydrolysis is necessary for the “power stroke” that drives transport. We suggest that the transition state energy for the conformational changes that form the E·S complex is coupled to the transport substrate site to do mechanical work.