Mutagenesis of the a subunit of the F1F0-ATP synthase from Escherichia coli in the region of Asn-192.
Site-directed cassette mutagenesis was used to generate a series of amino acid substitutions in the a subunit of the Escherichia coli F1F0-ATP synthase. The following substitutions for Asn-192 were analyzed and shown to inhibit partially the ATP-dependent proton translocation without disrupting F1-F0 interactions: Leu, Val, Pro, Ser, Thr, and Arg. A group of multiple substitutions at residues Gln-181, Asn-184, and His-185 had no significant effect on ATP synthase function, as judged by growth yields, or by assays of ATP-dependent proton translocation, indicating that this region of the a subunit is not involved in function. Three double mutants were constructed in order to assess the independence of residues Asn-192, Glu-196, and Asn-214. Results of proton translocation assays of membranes from cells containing these double mutations are consistent with the interpretation that each of these residues is involved with proton movement, and that residues Asn-192 and Glu-196 may be coupled. Finally, the relationship between the mechanism of proton translocation by the E. coli ATP synthase and the chloroplast enzyme was probed by constructing variants of the E. coli a subunit containing several features of homologous chloroplast proteins. It was determined that these chloroplast features, in the region of Glu-196 of the E. coli a subunit,, were detrimental to ATP synthase function.
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Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275-0376.
Lee
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Curtis
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Nguyen
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D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D001216:Asparagine; D006180:Proton-Translocating ATPases