Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein.
ATPase activity associated with P-glycoprotein (Pgp) is characterized by three drug-dependent phases: basal (no drug), drug-activated, and drug-inhibited. To understand the communication between drug-binding sites and ATP hydrolytic sites, we performed steady-state thermodynamic analyses of ATP hydrolysis in the presence and absence of transport substrates. We used purified human Pgp (ABCB1, MDR1) expressed in Saccharomyces cerevisiae (Figler, R. A., Omote, H., Nakamoto, R. K., and Al-Shawi, M. K. (2000) Arch. Biochem. Biophys. 376, 34-46) as well as Chinese hamster Pgp (PGP1). Between 23 and 35 degrees C, we obtained linear Arrhenius relationships for the turnover rate of hydrolysis of saturating MgATP in the presence of saturating drug concentrations (kcat), from which we calculated the intrinsic enthalpic, entropic, and free energy terms for the rate-limiting transition states. Linearity of the Arrhenius plots indicated that the same rate-limiting step was being measured over the temperature range employed. Using linear free energy analysis, two distinct transition states were found: one associated with uncoupled basal activity and the other with coupled drug transport activity. We concluded that basal ATPase activity associated with Pgp is not a consequence of transport of an endogenous lipid or other endogenous substrates. Rather, it is an intrinsic mechanistic property of the enzyme. We also found that rapidly transported substrates bound tighter to the transition state and required fewer conformational alterations by the enzyme to achieve the coupling transition state. The overall rate-limiting step of Pgp during transport is a carrier reorientation step. Furthermore, Pgp is optimized to transport drugs out of cells at high rates at the expense of coupling efficiency. The drug inhibition phase was associated with low affinity drug-binding sites. These results are consistent with an expanded version of the alternating catalytic site drug transport model (Senior, A. E., Al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett. 377, 285-289). A new kinetic model of drug transport is presented.
Al-Shawi
Marwan K
MK
Department of Molecular Physiology and Biological Physics, University of Virginia Health System, Charlottesville, Virginia 22908-0736, USA. ma9a@virginia.edu
Polar
Mark K
MK
Omote
Hiroshi
H
Figler
Robert A
RA
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002384:Catalysis; D003078:Colchicine / Q000494:pharmacology; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D019277:Entropy; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008954:Models, Biological; D000069463:Olive Oil; D010938:Plant Oils / Q000737:chemistry; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008055:Lipids; D000069463:Olive Oil; D010938:Plant Oils; D000255:Adenosine Triphosphate; D014700:Verapamil; D000251:Adenosine Triphosphatases; D003078:Colchicine