Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein

In the nucleotide-binding domains (NBDs) of ABC transporters, such as mouse Mdr3 P-glycoprotein (P-gp), an invariant carboxylate residue (E552 in NBD1; E1197 in NBD2) immediately follows the Walker B motif (hyd(4)DE/D). Removal of the negative charge in mutants E552Q and E1197Q abolishes drug-stimulated ATPase activity measured by P-i release. Surprisingly, drug-stimulated trapping of 8-azido-[alpha-P-32]ATP is still observed in the mutants in both the presence and absence of the transition-state analogue vanadate (V-i), and ADP can be recovered from the trapped enzymes. The E552Q and E1197Q mutants show characteristics similar to those of the wild-type (WT) enzyme with respect to 8-azido-[alpha-P-32]ATP binding and 8-azido-[alpha-P-32]nucleotide trapping, with the latter being both Mg2+ and temperature dependent. Importantly, drug-stimulated nucleotide trapping in E552Q is stimulated by V-i and resembles the WT enzyme, while it is almost completely V-i insensitive in E1197Q. Similar nucleotide trapping properties are observed when aluminum fluoride or beryllium fluoride is used as an alternate transition-state analogue. Partial proteolytic cleavage of photolabeled enzymes indicates that, in the absence of V-i, nucleotide trapping occurs exclusively at the mutant NBD, whereas in the presence of V-i, nucleotide trapping occurs at both NBDs. Together, these results suggest that there is single-site turnover occurring in the E552Q and E1197Q mutants and that ADP release from the mutant site, or another catalytic step, is impaired in these mutants. Furthermore, our results support a model in which the two NBDs of P-gp are not functionally equivalent.