How to study Pgp catalysis (Dey, 1997). Pgp contains two nucleotide binding sites (NBS), both of which are capable of binding ATP. Stoichiometric studies indicate that 2 ATP are required per Pgp catalytic cycle. Determining how those ATP are utilized is aided using vanadate (Vi), which traps nucleotides by mimicking Pi. When Vi is incubated with cell lines expressing Pgp, ATP hydrolysis is arrested. Vi appears to inhibit Pgp and delay its transition through the catalytic cycle by trapping ADP at the catalytic site (Figure 10). This information can be used to study the mechanistic details of the Pgp catalytic cycle.

Figure 10

ADP and substrate binding (Zerr, 2000). Vi-induced trapping of Pgp revealed a correlation between disassociation of labeled ADP and recovery of substrate binding. Cell lines expressing Pgp were incubated with Vi and a labeled ADP. Excess Vi and ADP were then removed and ATP was added. Binding ability for a substrate Pgp normally binds well, IAAP, as well as the rate of ADP disassociation were measured. A direct relationship between ADP disassociation and IAAP binding was determined (Figure 11A).

Figure 11 (figure taken from Zerr, 2001)

While other intermediate steps may be involved, this evidence indicates that the release of a nucleotide stimulates substrate binding. This represents one distinct step in the catalytic mechanism of Pgp action.

ATP hydrolyis (Zerr, 2000). Vi trapping can also be used to specify the nature of the reaction step which permits substrate binding. If the experiment mentioned above is repeated using nonhyrolyzable ATP (AMPPNP), Pgp cannot bind substrate, indicating that the hydrolysis of an ATP molecule permits substrate binding (Figure 12).

Figure 12 (figure taken from Zerr, 2000)

A conformational change must occur in Pgp upon ATP hydrolysis which permits substrate binding. The stoichiometric requirement for two ATP/catalytic cycle is explained by an additional ATP hydrolysis step that is necessary to return Pgp to its original conformation.