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The invariant carboxylate residue which follows the Walker B motif (hyd4DE⁄D) in the nucleotide-binding domains (NBDs) of ATP-binding cassette transporters is thought to be involved in the hydrolysis of the c-phosphate of MgATP, either by activating the attacking water molecule or by promoting substrate-assisted catalysis. | ễFEBS Journal Investigating the role of the invariant carboxylate residues E552 and E1197 in the catalytic activity of Abcbla mouse Mdr3 Isabelle Carrier and Philippe Gros Department of Biochemistry and McGillCancer Centre McGillUniversity Montreal Canada Keywords ABC transporter Abcb1a ATP hydrolysis catalytic mechanism nucleotide-binding domain Correspondence P. Gros Department of Biochemistry and McGillCancer Centre McGillUniversity McIntyre MedicalSciences Building Room 907 3655 Sir William Osler Drive Montreal Quebec H3G 1Y 6 Canada Fax 1 514 398 2603 Tel 1 514 398 7291 E-mail philippe.gros@mcgill.ca Received 6 February 2008 revised 26 March 2008 accepted 24 April 2008 doi 10.1111 j.1742-4658.2008.06479.x The invariant carboxylate residue which follows the Walker B motif hyd4DE D in the nucleotide-binding domains NBDs of ATP-binding cassette transporters is thought to be involved in the hydrolysis of the y-phosphate of MgATP either by activating the attacking water molecule or by promoting substrate-assisted catalysis. In Abcbla this invariant carboxylate residue corresponds to E552 in NBD1 and E1197 in NBD2. To further characterize the role of these residues in catalysis we created in Abcbla the single-site mutants E552D N and A in NBD1 and E1197D N and A in NBD2 as well as the double-mutant E552Q E1197Q. In addition we created mutants in which the Walker A K fi R mutation known to abolish ATPase activity was introduced in the non-mutant NBD of E552Q and E1197Q. ATPase activity binding affinity and trapping properties were tested for each Abcbla variant. The results suggest that the length of the invariant carboxylate residue is important for the catalytic activity whereas the charge of the side chain is critical for full turnover to occur. Moreover in the double-mutants where the K fi R mutation is introduced in the wild-type NBD of the E fi Q mutants single-site turnover is observed especially when NBD2 can undergo y-Pi cleavage. The results further support
