The conserved glutamate residue adjacent to the Walker-B motif is the catalytic base for ATP hydrolysis in the ATP-binding cassette transporter BmrA.

摘要: ATP-binding cassette (ABC) proteins constitute one of the widest families in all organisms, whose P-glycoprotein involved resistance cancer cells to chemotherapy is an archetype member. Although three-dimensional structures several nucleotide-binding domains ABC are now available, catalytic mechanism triggering functioning these still remains elusive. In particular, it has been postulated that ATP hydrolysis proceeds via acid-base catalyzed by Glu residue adjacent Walker-B motif (Geourjon, C., Orelle, Steinfels, E., Blanchet, Deleage, G., Di Pietro, A., and Jault, J. M. (2001) Trends Biochem. Sci. 26, 539–544), but involvement such as base transporters was recently questioned (Sauna, Z. Muller, M., Peng, X. H., Ambudkar, S. V. (2002) Biochemistry, 41, 13989–14000). The equivalent glutamate (Glu504) a half-ABC transporter multidrug Bacillus subtilis, BmrA (formerly known YvcC), therefore mutated Asp, Ala, Gln, Ser, Cys residues. All mutants were fully devoid ATPase activity, yet they showed high level vanadate-independent trapping 8-N3-α-32P-labeled nucleotide(s), following preincubation with 8-N3-[α-32P]ATP. However, contrast wild-type enzyme, use 8-N3-[γ-32P]ATP unequivocally trapped exclusively triphosphate form analogue, suggesting not able perform even single hydrolytic turnover. These results demonstrate Glu504 for BmrA, proposed residues other play same role.