The conserved glutamate residue adjacent to the Walker-B motif is the catalytic base for ATP hydrolysis in the ATP-binding cassette transporter BmrA.
ATP-binding cassette (ABC) proteins constitute one of the widest families in all organisms, whose P-glycoprotein involved in resistance of cancer cells to chemotherapy is an archetype member. Although three-dimensional structures of several nucleotide-binding domains of ABC proteins are now available, the catalytic mechanism triggering the functioning of these proteins still remains elusive. In particular, it has been postulated that ATP hydrolysis proceeds via an acid-base mechanism catalyzed by the Glu residue adjacent to the Walker-B motif (Geourjon, C., Orelle, C., Steinfels, E., Blanchet, C., Deléage, G., Di Pietro, A., and Jault, J. M. (2001) Trends Biochem. Sci. 26, 539-544), but the involvement of such residue as the catalytic base in ABC transporters was recently questioned (Sauna, Z. E., Muller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry, 41, 13989-14000). The equivalent glutamate residue (Glu504) of a half-ABC transporter involved in multidrug resistance in Bacillus subtilis, BmrA (formerly known as YvcC), was therefore mutated to Asp, Ala, Gln, Ser, and Cys residues. All these mutants were fully devoid of ATPase activity, yet they showed a high level of vanadate-independent trapping of 8-N3-alpha-32P-labeled nucleotide(s), following preincubation with 8-N3-[alpha-32P]ATP. However, and in contrast to the wild-type enzyme, the use of 8-N3-[gamma-32P]ATP unequivocally showed that all the mutants trapped exclusively the triphosphate form of the analogue, suggesting that they were not able to perform even a single hydrolytic turnover. These results demonstrate that Glu504 is the catalytic base for ATP hydrolysis in BmrA, and it is proposed that equivalent glutamate residues in other ABC transporters play the same role.
Orelle
Cédric
C
Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-UCBL1 and IFR 128, 7 passage du Vercors, 69367 Lyon Cedex 07, France.
Dalmas
Olivier
O
Gros
Philippe
P
Di Pietro
Attilio
A
Jault
Jean-Michel
JM
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D002384:Catalysis; D017124:Conserved Sequence; D024881:Drug Resistance, Bacterial; D018698:Glutamic Acid; D006868:Hydrolysis; D016415:Sequence Alignment
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D018698:Glutamic Acid; D000255:Adenosine Triphosphate