Marwan K. Al-Shawi

THE CATALYTIC CYCLE OF P-GLYCOPROTEIN

P‐glycoprotein is a plasma‐membrane glycoprotein which confers multidrug‐resistance on cells and displays ATP‐driven drug‐pumping in vitro. It contains two nucleotide‐binding domains, and its structure places it in the ‘ABC transporter’ family. We review recent evidence that both nucleotide‐sites bind and hydrolyse Mg‐ATP. The two catalytic sites interact strongly. A minimal scheme for the MgATP hydrolysis reaction is presented. An alternating catalytic sites scheme is proposed, in which drug transport is coupled to relaxation of a high‐energy catalytic site conformation generated by the hydrolysis step. Other ABC transporters may show similar catalytic features.

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 °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.

The rotary mechanism of the ATP synthase

The F0F1 ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous F0 sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium.

ATP hydrolysis by multidrug-resistance protein from Chinese hamster ovary cells

ATPase activity of multidrug-resistance protein (P-glycoprotein, Pgp) from Chinese hamster ovary cells was studied. Catalytic characteristics were established for Pgp both in its natural plasma membrane environment and in purified reconstituted protein. Generally the two preparations of Pgp behaved similarly, and demonstrated low affinity for MgATP, low nucleotide specificity, preference for Mg-nucleotide, and pH optimum near 7.5. A high-affinity binding site involved in catalysis was not apparent. Effective covalent inactivators were NBD-C1, NEM, 8-azido-ATP, and 2-azido-ATP. DCCD, FITC, and pyridoxal phosphate were only weakly inhibitory. Lipid composition was found to affect the degree of drug stimulation of ATPase in purified reconstituted Pgp, suggesting that the lipid environment affects coupling between drug-binding and catalytic sites, and that Pgp expressed in different tissues could show different functional characteristics.

Energy coupling, turnover, and stability of the F0F1 ATP synthase are dependent on the energy of interaction between gamma and beta subunits.

Replacement of the F0F1 ATP synthase γ subunit Met-23 with Lys (γM23K) perturbs coupling efficiency between transport and catalysis (Shin, K., Nakamoto, R. K., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267, 20835-20839). We demonstrate here that the γM23K mutation causes altered interactions between subunits. Binding of δ or ε subunits stabilizes the α3β3γ complex, which becomes destabilized by the mutation. Significantly, the inhibition of F1 ATP hydrolysis by the ε subunit is no longer relieved when the γM23K mutant F1 is bound to F0. Steady state Arrhenius analysis reveals that the γM23K enzyme has increased activation energies for the catalytic transition state. These results suggest that the mutation causes the formation of additional bonds within the enzyme that must be broken in order to achieve the transition state. Based on the x-ray crystallographic structure of Abrahams et al. (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the additional bond is likely due to γM23K forming an ionized hydrogen bond with one of the βGlu-381 residues. Two second site mutations, γQ269R and γR242C, suppress the effects of γM23K and decrease activation energies for the γM23K enzyme. We conclude that γM23K is an added function mutation that increases the energy of interaction between γ and β subunits. The additional interaction perturbs transmission of conformational information such that ε inhibition of ATPase activity is not relieved and coupling efficiency is lowered.

The ATP synthase gamma subunit. Suppressor mutagenesis reveals three helical regions involved in energy coupling.

A role in coupling proton transport to catalysis of ATP synthesis has been demonstrated for the Escherichia coli F0F1 ATP synthase γ subunit. Previously, functional interactions between the terminal regions that were important for coupling were shown by finding several mutations in the carboxyl-terminal region of the γ subunit (involving residues at positions 242 and 269-280) that restored efficient coupling to the mutation, γMet-23 → Lys (Nakamoto, R. K., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 867-872). In this study, we used suppressor mutagenesis to establish that the terminal regions can be separated into three interacting segments. Second-site mutations that cause pseudo reversion of the primary mutations, γGln-269 → Glu or γThr-273 → Val, map to an amino-terminal segment with changes at residues 18, 34, and 35, and to a segment near the carboxyl terminus with changes at residues 236, 238, 242, and 246. Each second-site mutation suppressed the effects of both γGln-269 → Glu and γThr-273 → Val, and restored efficient coupling to enzyme complexes containing either of the primary mutations. Mapping of these residues in the recently reported x-ray crystallographic structure of the F1 complex (Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E.(1994) Nature 370, 621-628), reveals that the second-site mutations do not directly interact with γGln-269 and γThr-273 and that the effect of suppression occurs at a distance. We propose that the three γ subunit segments defined by suppressor mutagenesis, residues γ18-35, γ236-246, and γ269-280, constitute a domain that is critical for both catalytic function and energy coupling.

Single Molecule Behavior of Inhibited and Active States of Escherichia coli ATP Synthase F1 Rotation

ATP hydrolysis-dependent rotation of the F1 sector of the ATP synthase is a successive cycle of catalytic dwells (∼0.2 ms at 24 °C) and 120° rotation steps (∼0.6 ms) when observed under Vmax conditions using a low viscous drag 60-nm bead attached to the γ subunit (Sekiya, M., Nakamoto, R. K., Al-Shawi, M. K., Nakanishi-Matsui, M., and Futai, M. (2009) J. Biol. Chem. 284, 22401–22410). During the normal course of observation, the γ subunit pauses in a stochastic manner to a catalytically inhibited state that averages ∼1 s in duration. The rotation behavior with adenosine 5′-O-(3-thiotriphosphate) as the substrate or at a low ATP concentration (4 μm) indicates that the rotation is inhibited at the catalytic dwell when the bound ATP undergoes reversible hydrolysis/synthesis. The temperature dependence of rotation shows that F1 requires ∼2-fold higher activation energy for the transition from the active to the inhibited state compared with that for normal steady-state rotation during the active state. Addition of superstoichiometric ϵ subunit, the inhibitor of F1-ATPase, decreases the rotation rate and at the same time increases the duration time of the inhibited state. Arrhenius analysis shows that the ϵ subunit has little effect on the transition between active and inhibited states. Rather, the ϵ subunit confers lower activation energy of steady-state rotation. These results suggest that the ϵ subunit plays a role in guiding the enzyme through the proper and efficient catalytic and transport rotational pathway but does not influence the transition to the inhibited state.

Catalytic and transport cycles of ABC exporters.

ABC (ATP-binding cassette) transporters are arguably the most important family of ATP-driven transporters in biology. Despite considerable effort and advances in determining the structures and physiology of these transporters, their fundamental molecular mechanisms remain elusive and highly controversial. How does ATP hydrolysis by ABC transporters drive their transport function? Part of the problem in answering this question appears to be a perceived need to formulate a universal mechanism. Although it has been generally hoped and assumed that the whole superfamily of ABC transporters would exhibit similar conserved mechanisms, this is proving not to be the case. Structural considerations alone suggest that there are three overall types of coupling mechanisms related to ABC exporters, small ABC importers and large ABC importers. Biochemical and biophysical characterization leads us to the conclusion that, even within these three classes, the catalytic and transport mechanisms are not fully conserved, but continue to evolve. ABC transporters also exhibit unusual characteristics not observed in other primary transporters, such as uncoupled basal ATPase activity, that severely complicate mechanistic studies by established methods. In this chapter, I review these issues as related to ABC exporters in particular. A consensus view has emerged that ABC exporters follow alternating-access switch transport mechanisms. However, some biochemical data suggest that alternating catalytic site transport mechanisms are more appropriate for fully symmetrical ABC exporters. Heterodimeric and asymmetrical ABC exporters appear to conform to simple alternating-access-type mechanisms.

Molecular mechanisms of rotational catalysis in the F0F1 ATP synthase

Rotation of the F(0)F(1) ATP synthase gamma subunit drives each of the three catalytic sites through their reaction pathways. The enzyme completes three cycles and synthesizes or hydrolyzes three ATP for each 360 degrees rotation of the gamma subunit. Mutagenesis studies have yielded considerable information on the roles of interactions between the rotor gamma subunit and the catalytic beta subunits. Amino acid substitutions, such as replacement of the conserved gammaMet-23 by Lys, cause altered interactions between gamma and beta subunits that have dramatic effects on the transition state of the steady state ATP synthesis and hydrolysis reactions. The mutations also perturb transmission of specific conformational information between subunits which is important for efficient conversion of energy between rotation and catalysis, and render the coupling between catalysis and transport inefficient. Amino acid replacements in the transport domain also affect the steady state catalytic transition state indicating that rotation is involved in coupling to transport.

ATPASE ACTIVITY OF CHINESE HAMSTER P-GLYCOPROTEIN

We have developed two defined experimental systems for biochemical investigation of P-glycoprotein, namely, plasma membranes highly enriched in Pgp, obtained from the CR1R12 Chinese hamster ovary cell line, and pure, reconstituted Pgp, obtained by solubilization of Pgp from CR1R12 plasma membranes, Reactive Red 120 chromatography, and reconstitution in liposomes. Studies of the ATPase catalytic mechanism by kinetic methods and covalent inactivation have been greatly facilitated by the availability of these experimental systems. The technique of vanadate trapping of nucleotide has been particularly useful. As a result of these studies, we now have explicit, testable, proposals for (1) the normal catalytic pathway of ATP hydrolysis, (2) a postulated alternating catalytic site cycle, and (3) coupling of ATP hydrolysis to drug transport. The experimental methods described here should prove valuable for future studies of Pgp and of ABC transporters in general.