Mutations altering aspartyl-61 of the omega subunit (uncE protein) of Escherichia coli H+ -ATPase differ in effect on coupled ATP hydrolysis.
Mutations in the H+-translocating ATPase complex (F1F0) of Escherichia coli have been described in which aspartyl-61 of the omega subunit ( uncE protein) is substituted by either glycine ( uncE105 ) or asparagine ( uncE107 ). Either substitution blocks the H+-translocation activity of the F0 sector of the complex. Here we report a difference in the effects of the two substitutions on the coupled ATPase activity of F1 bound to F0. Wild-type F1 was bound to the F0 of either mutant with affinities comparable to wild-type. The ATPase activity of F1 bound to uncE107 F0 was inhibited by 50%, whereas that bound to uncE105 F0 was not inhibited. Complementation studies with a pBR322-derived plasmid that carried the E gene of the unc operon only indicated that a single mutation in the host strain was responsible for the respective phenotypes. In mutants complemented by the uncE + plasmid, restoration of wild-type biochemical properties was only partial and may be attributed to a mixing of wild-type and mutant omega subunits in a hybrid F0 complex. The activity of membrane-bound F1 was less inhibited in the uncE +/ uncE107 hybrid. Paradoxically, complementation of uncE105 by the uncE + plasmid resulted in substantial inhibition of the activity of membrane-bound F1. The results indicate that a glycine-versus-asparagine substitution for aspartyl-61 must lead to altered conformations of omega and that these differences in conformation are important in the coupling between the F0 and F1 sectors of the complex.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D046911:Macromolecular Substances; D009154:Mutation; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000235:genetics*
D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D004262:DNA Restriction Enzymes; D006180:Proton-Translocating ATPases