A phenylalanine for serine substitution in the beta subunit of Escherichia coli F1-ATPase affects dependence of its activity on divalent cations.
A mutant (KF11) of Escherichia coli H+-translocating ATPase (F1-F0) has a single point mutation in the beta subunit of F1 that has lost 90% of its Mg2+-dependent ATPase activity (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., and Futai, M. (1980) J. Biochem. (Tokyo) 88, 695-703). The mutation was mapped at about the 500th nucleotide residue from the 5' end of the beta subunit gene by a genetic recombination test on the physical map of the cistron coding for the beta subunit. The mutant allele of KF11 (uncD11) was cloned on a hybrid plasmid (pKF11) via DNA isolated from a lambda uncD11 transducing phage. Restriction fragments of pKF11 containing the estimated mutation site were subjected to polyacrylamide gel electrophoresis under conditions where strands were separated into single strands. The two strands of a DNA segment, which was shown to carry an altered base, showed anomalous migration compared with those from the wild-type fragment. The results confirmed the result of mapping of the altered site by genetic tests. On the basis of these results, the nucleotide sequence of the mutated gene was determined, and a single base change of the 524th cytosine to thymine resulting in a phenylalanine for serine substitution at residue 174 of the beta subunit was found. This result, together with results on the altered properties of F1 from KF11 reported previously, indicates that residue 174 is essential for the Mg2+-dependent ATPase activity of F1 but not for the Ca2+-dependent ATPase activity.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D009154:Mutation; D010649:Phenylalanine; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D012694:Serine
D002413:Cations, Divalent; D046911:Macromolecular Substances; D012694:Serine; D010649:Phenylalanine; D004262:DNA Restriction Enzymes; D006180:Proton-Translocating ATPases