The Escherichia coli F 1 -ATPase mutant βTyr-297 → Cys: functional studies and asymmetry of the enzyme under various nucleotide conditions based on reaction of the introduced Cys with N-Ethylmaleimide (cas 128-53-0) and 7-chloro-4-nitrobenzofurazan
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Add time:09/30/2019 Source:infona.pl
Conversion of residue βTyr-297 of the Escherichia coli F 1 -ATPase (ECF 1 ) to a Cys in the mutant βY297C led to impaired oxidative phosphorylation based on growth curves. The ATPase activity of ECF 1 isolated from the mutant βY297C 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 βCys-297. The mutation βTyr-297 → Cys had no effect on nucleotide binding in studies with the fluorescent analoglin -benzo-ADP. The asymmetry of ECF 1 was investigated in the mutants βY297C and βY297C:E381C/εS108C by examining the relative reactivity of Cys-297 in the three copies of the β 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 β f r e e , which is also the site most reactive to NEM, β ε is the second site which reacts with NbfCl or NEM, while the third site, β γ , is poorly reactive to either reagent.
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