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N ‐Ethylmaleimide‐sensitive mutant (βVal‐153→Cys) Escherichia coli F1‐ATPase: Cross‐linking of the mutant β subunit with the α subunit
Author(s) -
Iwamoto Atsuko,
Orita-Saita Yuji,
Maeda Masatomo,
Futai Masamitsu
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)00963-5
Subject(s) - mutant , protein subunit , n ethylmaleimide , escherichia coli , cooperativity , enzyme , atpase , biochemistry , biology , microbiology and biotechnology , mutation , chemistry , gene
A β subunit mutation, βVal‐153→Cys, in the glycine‐rich sequence (phosphate‐binding loop) of Escherichia coli F 1 was constructed. Like vacuolar‐type ATPase, the mutant enzyme was inhibited by N ‐ethylmaleimide (NEM) and labeled with [ 14 C]NEM. The inhibition and labeling were prevented by ATP. m ‐Maleimidobenzoyl‐ N ‐hydroxysuccinimide (MBS) (3 μM) almost completely inhibited the mutant enzyme, and cross‐linked one pair of α and β subunits. These results suggest that the interaction of the domain near βVal‐153 with the α subunit is essential for catalytic cooperativity of the enzyme and that βVal‐153 is within 10 Å of the α subunit.