, 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.

A simple and rapid procedure is described for purification of P-glycoprotein (Pgp) from a multidrug-resistant Chinese hamster ovary cell line (CR1R12) in which the plasma membranes are highly enriched in Pgp (Al-Shawi, M.K., Senior A.E. (1993) J. Biol. Chem, 268, 4197-4206). The procedure consisted of octylglucoside solubilization of Pgp from plasma membranes and chromatography on Reactive Red 120 agarose. The purified Pgp displayed substantial verapamil-stimulated MgATPase activity (kcat = 9.2 s-1, KM(MgATP) = 0.8 mM). A range of other compounds known to interact with Pgp in whole cells also stimulated the MgATPase activity. Catalytic activity in presence of verapamil was characterized in terms of pH dependence, magnesium versus calcium specificity, kinetic parameters, nucleotide specificity, and inhibitors. There was potent inactivation of MgATPase activity by NEM and NBD-Cl, which was diminished greatly by MgATP protection. Vanadate was also an effective inhibitor. Predominantly, the catalytic features seen resembled those reported previously for the plasma membrane-bound form of Pgp. The catalytic nucleotide-binding sites are therefore preserved in their native folded conformation in the purified Pgp preparation.

The F O F 1 ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous F O 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. KeywordsATP synthase; kinetic mechanism; rotation; transport Like many transporters, the F O F 1 ATP synthase (or F-type ATPase) has been a fascinating subject for the study of a complex membrane-associated process. The ATP synthase is a critically important activity that carries out synthesis of ATP from ADP and Pi driven by a proton motive force, Δµ H+ , or sodium motive force, Δµ Na+ . This final step of oxidative or photo-phosphorylation provides the vast majority of ATP in the cell. The proton or sodium motive force is also needed to power other membrane processes such as secondary transporters or in the case of bacteria, flagellum rotation. In anaerobic conditions, facultative bacteria use the ATP synthase as an ATP-driven H + or Na + pump to generate the Δµ H+ , or Δµ Na+ (see [1] for a textbook review.) The F O F 1 complex is nearly ubiquitous in the cell membranes of eubacteria, in the thylakoid membrane of chloroplasts, and the inner membrane of mitochondria. The transporter has remained structurally and mechanistically conserved, except for a few additional domains or subunits in mitochondria, which may play roles in regulation or assembly.Many years of innovative biochemical, genetic, kinetic, and thermodynamic studies led to the first structural solution of the catalytic F 1 portion of the complex by Walker, Leslie and coworkers [2] in 1994. This landmark structure provided critical information on the catalytic portion of the complex but the subunit arrangement of much of the rest of the complex was still not elucidated. The partial F 1 structure, which at the time was the largest asymmetric unit solved, provided the impetus and the structural information needed to test the notion that the

Replacement of the F 0 F 1 ATP synthase ␥ subunit Met-23 with Lys (␥M23K) 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 ␥M23K mutation causes altered interactions between subunits. Binding of ␦ or ⑀ subunits stabilizes the ␣ 3 ␤ 3 ␥ complex, which becomes destabilized by the mutation. Significantly, the inhibition of F 1 ATP hydrolysis by the ⑀ subunit is no longer relieved when the ␥M23K mutant F 1 is bound to F 0 . Steady state Arrhenius analysis reveals that the ␥M23K 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 ␥M23K forming an ionized hydrogen bond with one of the ␤Glu-381 residues. Two second site mutations, ␥Q269R and ␥R242C, suppress the effects of ␥M23K and decrease activation energies for the ␥M23K enzyme. We conclude that ␥M23K is an added function mutation that increases the energy of interaction between ␥ and ␤ subunits. The additional interaction perturbs transmission of conformational information such that ⑀ inhibition of ATPase activity is not relieved and coupling efficiency is lowered.The F 0 F 1 ATP synthase links two disparate functions: transport of protons across a membrane and catalysis of ATP synthesis or hydrolysis (for reviews see Refs. 1-5). The fully cooperative mechanism of ATP hydrolysis requires a minimum of three different subunits in a complex containing ␣ 3 ␤ 3 ␥ 1 . The transport mechanism is most likely assembled from F 0 sector subunits. In the Escherichia coli complex, transport requires three different membrane-spanning subunits, a 1 b 2 c ϳ10 (6). In addition, two more soluble subunits, ␦ and ⑀, are needed to reconstitute catalytic and transport sectors so that they are coupled to carry out ATP-driven proton pumping or ⌬ Hϩ -driven ATP synthesis (7-10).Catalysis and transport mechanisms most likely communicate indirectly through a series of conformational and electrostatic interactions. Conformational changes relevant to the catalytic state of the enzyme or the presence of a ⌬ H ϩ have been detected by several methods including altered cross-linking patterns, protease susceptibility, environmentally sensitive fluorescent probes, accessibility of epitopes, x-ray diffraction, cryoelectron microscopy, and spectroscopic analyses (reviewed in Refs. 11 and 12). High resolution structural information based on crystals of the bovine mitochondrial F 1 has also provided a great deal of information about possible subunit interactions that may be involved in linking transport and catalysis (13).Mutagenic analysis has also yielded important information about the coupl...

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