title: Replacement of arginine 246 by histidine in the beta subunit of Escherichia coli H+-ATPase resulted in loss of multi-site ATPase activity.
abstract: A mutant strain KF43 of Escherichia coli defective in the beta subunit of H+-translocating ATPase (F0F1) was examined. In this mutant, replacement of Arg246 by His was identified by DNA sequencing of the mutant gene and confirmed by tryptic peptide mapping. The mutant F1-ATPase was defective in multi-site hydrolysis of ATP but was active in uni-site hydrolysis. Studies on the kinetics of uni-site hydrolysis indicated that the k1 (rate of ATP binding) was similar to that of the wild-type, but the k-1 (rate of release of ATP) could not be measured. The mutant enzyme had a k3 (rate of release of inorganic phosphate) about 15-fold higher than that of the wild-type and showed 3 orders of magnitude lower promotion from uni- to multi-site catalysis. These results suggest that Arg246 or the region in its vicinity is important in multi-site hydrolysis of ATP and is also related to the binding of inorganic phosphate. Reconstitution experiments using isolated subunits suggested that hybrid enzymes (alpha beta gamma complexes) carrying both the mutant and wild-type beta subunits were inactive in multi-site hydrolysis of ATP, supporting the notion that three intact beta subunits are required for activity of the F1 molecule.
authors.forename: T
authors.initials: T
authors.lastname: Noumi
authors.forename: M
authors.initials: M
authors.lastname: Taniai
authors.forename: H
authors.initials: H
authors.lastname: Kanazawa
authors.forename: M
authors.initials: M
authors.lastname: Futai
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine; D007700:Kinetics; D009154:Mutation; D010710:Phosphates / Q000378:metabolism; D055503:Protein Multimerization; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D004269:DNA, Bacterial; D010710:Phosphates; D006639:Histidine; D000255:Adenosine Triphosphate; D001120:Arginine; D006180:Proton-Translocating ATPases