Site-directed mutagenesis of the conserved beta subunit tyrosine 331 of Escherichia coli ATP synthase yields catalytically active enzymes.
The ATP synthases of eubacteria and eukaryotes possess a conserved tyrosine (beta 331) that is labeled by ATP analogs and is believed to be at the catalytic site. In this report, this tyrosine was replaced by Phe, Ser, Cys, Gly, and Ala in an attempt to determine its role in catalysis. Each of the beta 331 mutant strains assembled an ATP synthase. Membranes from the beta 331-Ser, -Cys, -Ala, or -Gly strains showed strongly attenuated ATP hydrolysis and ATP-driven proton-pumping activities. The beta 331-Phe membranes showed nearly normal ATPase and functional proton pumping. A new purification procedure yielding highly active unc+ F1 (ATPase rates greater than 1000 s-1) allowed rapid isolation of soluble F1-ATPases. Kinetic analyses of purified enzymes confirmed that the structural and functional properties of beta 331-Tyr can be substituted by Phe but not effectively by Ser, Cys, Ala, or Gly. Since all of the beta 331 mutant enzymes catalyzed measurable ATP hydrolysis, it is clear that beta 331-Tyr is not directly involved in the bond making-breaking steps of catalysis. The ability of the beta 331-Phe enzyme to rapidly bind and hydrolyze ATP, and the results with other beta 331 mutant enzymes, suggests that a residue with an aromatic character is required at this position.
Wise
J G
JG
Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse 13210.
D000483:Alleles; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005798:Genes, Bacterial; D005838:Genotype; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D014443:Tyrosine
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D014443:Tyrosine; D006180:Proton-Translocating ATPases