Mechanistic complications. As more evidence accrues, the complexity of the Pgp catalytic cycle increases. Additional research has added to the available information concerning Pgp and nucleotide binding to show that while nucleotide binding does not directly affect Pgp/substrate interactions, ATP hydrolysis does result in a conformational change. This change results in a series of steps which lead to Pgp-catalyzed drug efflux.

Hydrolysis and Nucleotide Binding Sites (Sauna, 2001) As shown on the Nucleotide binding page, Vi-induced trapping and incubation with a labeled ATP has demonstrated that after the first hydrolysis event, Pgp substrate affinity is greatly reduced. The decrease in binding follows a concentration-dependent, single-phase exponential decay curve. The figure shown below integrates information about nucleotide binding, nucleotide hydrolysis, and substrate (IAAP) affinity. Stage I represents the first ATP binding. Stage II represents the hydrolyis of the first ATP. Stage II represents the binding of the second ATP while Stage IV represents the hydrolysis of the second ATP. As shown, substrate affinity is greatly reduced upon the hydrolysis of the first ATP and is not recovered until after the hydrolysis of the second ATP (Stages V and Va) (Figure 13).

Figure 13 (figure taken from Sauna, 2001)

In addition, a labeled nucleotide allowed for the distinction between ATP binding at the N-terminus and C-terminus region. No difference was seen between the two sites, indicating that during hydrolysis events ATP sites are selected randomly.

ADP disassociation (Sauna, 2001). To examine the role of hydrolyzed nucleotides in the Pgp catalytic cycle, labeled ATP proved very useful in monitoring Pgp action. Membranes containing Pgp were incubated with labeled ATP, washed, and then treated with non-labeled ATP. Results indicate that complete disassociation of the labeled nucleotides occurred in approximately 15 minutes. The disassociation was not affected by excess nucleotides or nonhydrolyzable ATP analogs, suggesting that ADP spontaneously disassociates during the Pgp catalytic cycle after it has been hydrolyzed.

Detailed Pgp catalytic model (Sauna, 2001) Combining experimental evidence led to the development of a more complex model of the Pgp catalytic mechanism (Figure 14).

I. Substrate (a hydrophobic drug) binds to the "On" substrate binding site and ATP binds to one of the nucleotide binding sites.

II. ATP is hydrolyzed and through a conformational change the substrate appears to move to the lower affinity binding site.

III. Pi is released from the hydrolzyed ATP and the substrate leaves.

IV. Hydrolyzed ADP disassociates from the complex. Pgp now has a low substrate affinity but a high nucleotide affinity.

V. A second molecule of ATP binds and is hydrolyzed.

VI. A second molecule of Pi is released, triggering a conformational change back to the original high substrate affinity state. The cycle repeats itself.

Figure 14 (figure taken from Sauna, 2001)