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Spatial precision of a catalytic carboxylate of F 1 ‐ATPase β subunit probed by introducing different carboxylate‐containing side chains
Author(s) -
Amano Toyoki,
Tozawa Kaeko,
Yoshida Masasuke,
Murakami Hideto
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)00588-5
Subject(s) - carboxylate , protein subunit , chemistry , catalysis , atpase , crystallography , stereochemistry , polymer chemistry , biochemistry , enzyme , gene
Combining mutation and chemical modification, we have introduced Asp, Gln, Cys, S ‐carboxymethylcysteine (Cax) and S ‐carbamoylmethylcysteine (Cam) into the positions of Glu 190 and Glu 201 of the β subunit of F 1 ‐ATPase from the thermophilic Bacillus PS3. The steady‐state ATPase activities of α 3 β 3 γ complexes containing these changed β subunits were 12% (E190Cax), 7% (E190D), 3% (E190Cam), <1% (E190C), <1% (E190Q), and 73% (E201D), 40% (E201Cax), 25% (E201C), 20% (E201Q), 4% (E201Cam), of that of the wild‐type α 3 β 3 γ complex. For the complexes containing E190C or E190Q, even the ability of single‐site catalysis was lost. Thus, the presence of a carboxylate at 190 (but not at 201) is absolutely required for catalysis and its spatial precision is very strict. Analysis of inactivation of the complexes by dicyclohexylcarbodiimide suggests that Glu 190 and Glu 201 are interacting in the F 1 ‐ATPase.