The Escherichia coli F1-ATPase mutant beta Tyr-297-->Cys: functional studies and asymmetry of the enzyme under various nucleotide conditions based on reaction of the introduced Cys with N-ethylmaleimide and 7-chloro-4-nitrobenzofurazan.
Conversion of residue beta Tyr-297 of the Escherichia coli F1-ATPase (ECF1) to a Cys in the mutant beta Y297C led to impaired oxidative phosphorylation based on growth curves. The ATPase activity of ECF1 isolated from the mutant beta Y297C was only 1% of wild-type activity, but the residual activity involves cooperative multi-site enzyme turnover based on inhibition by DCCD and azide. ATPase activity could be increased to 8%, and 13% of wild-type by reaction of the introduced Cys with N-ethyl maleimide (NEM), and 7-chloro-4-nitrobenzofurazan (NbfCl), respectively, suggesting that enzymatic function is improved by an increased hydrophobicity of residue beta Cys-297. The mutation beta Tyr-297-->Cys had no effect on nucleotide binding in studies with the fluorescent analog lin-benzo-ADP. The asymmetry of ECF1 was investigated in the mutants beta Y297C and beta Y297C:E381C/epsilon S108C by examining the relative reactivity of Cys-297 in the three copies of the beta subunit under different nucleotide binding conditions. In agreement with a previous study (Haughton, M.A. and Capaldi, R.A. (1995) J. Biol. Chem., 270, 20568-20574), the asymmetry was maintained under all nucleotide conditions. The NbfCl reaction site was found to be beta free, which is also the site most reactive to NEM, beta epsilon is the second site which reacts with NbfCl or NEM, while the third site, beta gamma, is poorly reactive to either reagent.
Haughton
M A
MA
Institute of Molecular Biology, University of Oregon, Eugene 97403-1229, USA.
Capaldi
R A
RA
D001572:Benzofurans / Q000494:pharmacology; D001665:Binding Sites; D003300:Copper / Q000494:pharmacology; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005033:Ethylmaleimide / Q000494:pharmacology; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D001572:Benzofurans; D003432:Cross-Linking Reagents; D009711:Nucleotides; D013439:Sulfhydryl Reagents; C043708:7-chloro-4-nitrobenzofuran; D003300:Copper; C028419:cuprous chloride; D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide