title: Conserved walker A Ser residues in the catalytic sites of P-glycoprotein are critical for catalysis and involved primarily at the transition state step.
abstract: P-glycoprotein mutants S430A/T and S1073A/T, affecting conserved Walker A Ser residues, were characterized to elucidate molecular roles of the Ser and functioning of the two P-glycoprotein catalytic sites. Results showed the Ser-OH is critical for MgATPase activity and formation of the normal transition state, although not for initial MgATP binding. Mutation to Ala in either catalytic site abolished MgATPase and transition state formation in both sites, whereas Thr mutants had similar MgATPase to wild-type. Trapping of 1 mol of MgADP/mol of P-glycoprotein by vanadate, shown here with pure protein, yielded full inhibition of ATPase. Thus, congruent with previous work, both sites must be intact and must interact for catalysis. Equivalent mutations (Ala or Thr) in the two catalytic sites had identical effects on a wide range of activities, emphasizing that the two catalytic sites function symmetrically. The role of the Ser-OH is to coordinate Mg(2+) in MgATP, but only at the stage of the transition state are its effects tangible. Initial substrate binding is apparently to an "open" catalytic site conformation, where the Ser-OH is dispensable. This changes to a "closed" conformation required to attain the transition state, in which the Ser-OH is a critical ligand. Formation of the latter conformation requires both sites; both sites may provide direct ligands to the transition state.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors.forename: I L
authors.initials: IL
authors.lastname: Urbatsch
authors.forename: K
authors.initials: K
authors.lastname: Gimi
authors.forename: S
authors.initials: S
authors.lastname: Wilke-Mounts
authors.forename: A E
authors.initials: AE
authors.lastname: Senior
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017124:Conserved Sequence; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008322:Mammals; D016297:Mutagenesis, Site-Directed; D010843:Pichia; D017354:Point Mutation; D012694:Serine; D014638:Vanadates / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D014638:Vanadates; D012694:Serine; D017301:Ca(2+) Mg(2+)-ATPase; D008274:Magnesium