Asymmetry of Escherichia coli F1-ATPase as a function of the interaction of alpha-beta subunit pairs with the gamma and epsilon subunits.
The asymmetry of Escherichia coli F1-ATPase (ECF1) has been explored in chemical modification experiments involving two mutant enzyme preparations. One mutant contains a cysteine (Cys) at position 149 of the beta subunit, along with conversion of a Val to Ala at residue 198 to suppress the deleterious effect of the Cys for Gly at 149 mutation (mutant beta G149C:V198A). The second mutant has these mutations and also Cys residues at positions 381 of beta and 108 of the epsilon subunit (mutant beta G149C:V198A:E381C/epsilon S108C). On CuCl2 treatment of this second mutant, there is cross-linking of one copy of the beta subunit to gamma via the Cys at 381, a second to the epsilon subunit (between beta Cys381 and epsilon Cys108), while the third beta subunit in the ECF1 complex is mostly free (some cross-linking to delta); thereby distinguishing the three beta subunits as beta gamma, beta epsilon, and beta free, respectively. Both mutants have ATPase activities similar to wild-type enzyme. Under all nucleotide conditions, including with essentially nucleotide-free enzyme, the three different beta subunits were found to react differently with N-ethylmaleimide (NEM) which reacts with Cys149, dicyclohexyl carbodiimide (DCCD) which reacts with Glu192, and 7-chloro-4-nitrobenzofurazan (NbfCl) which reacts with Tyr297. Thus, beta gamma reacted with DCCD but not NEM or NbfCl; beta free was reactive with all three reagents; beta epsilon reacted with NEM, but was poorly reactive to DCCD or NbfCl. There was a strong nucleotide dependence of the reaction of Cys149 in beta epsilon (but not in beta free) with NEM, indicative of the important role that the epsilon subunit plays in functioning of the enzyme.
Haughton
M A
MA
Institute of Molecular Biology, University of Oregon, Eugene 97403-1229, USA.
Capaldi
R A
RA
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001572:Benzofurans / Q000494:pharmacology; D001665:Binding Sites; D003300:Copper / Q000494:pharmacology; D003432:Cross-Linking Reagents; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D001572:Benzofurans; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D004024:Dicyclohexylcarbodiimide; D000244:Adenosine Diphosphate; C059027:4-chloro-7-nitrobenzofuran; D003300:Copper; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D005033:Ethylmaleimide; C029892:cupric chloride