Characterization of the catalytic cycle of ATP hydrolysis by human P-glycoprotein - The two ATP hydrolysis events in a single catalytic cycle are kinetically similar but affect different functional outcomes

P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression conf ers multidrug resistance to tumor cells by extruding amphipathic natural pr oduct cytotoxic drugs using the energy of ATP, An elucidation of the cataly tic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z, E,, and Ambudk ar, S, V. (2000) Proc. Natl, Acad Sci, U.S.A. 97, 2515-2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp, In this study we exploit the vanadate (Vi)induced transition state conforma tion of Pgp (Pgp ADP Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site, We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of n ucleotide for Pgp coincident with decreased substrate binding. Release of o ccluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two h ydrolysis events have different functional outcomes vis a vis the substrate , they show comparable t(1/2), for both incorporation and release of nucleo tide, and the affinities for [alpha-P-32]8-azido-ATP during Vi induced trap ping are identical. In addition, the incorporation of [alpha-P-32]8-azido-A DP in two ATP sites during both hydrolysis events is also similar. These da ta demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given ti me because of the conformational change in the catalytic site that drastica lly reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysi s of ATP hydrolysis and offer a mechanistic framework to elucidate events a t both the substrate- and nucleotide-binding sites in the catalytic cycle o f Pgp.