Spatial precision of a catalytic carboxylate of F1-ATPase beta subunit probed by introducing different carboxylate-containing side chains.
Combining mutation and chemical modification, we have introduced Asp, Gln, Cys, S-carboxymethylcysteine (Cax) and S-carbamoylmethylcysteine (Cam) into the positions of Glu190 and Glu201 of the beta subunit of F1-ATPase from the thermophilic Bacillus PS3. The steady-state ATPase activities of alpha 3 beta 3 gamma complexes containing these changed beta 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 that of the wild-type alpha 3 beta 3 gamma 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 Glu190 and Glu201 are interacting in the F1-ATPase.
Amano
T
T
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Yokohama, Japan.
Tozawa
K
K
Yoshida
M
M
Murakami
H
H
D000577:Amides / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism*; D002384:Catalysis; D004024:Dicyclohexylcarbodiimide; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid; D015394:Molecular Structure; D016296:Mutagenesis; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013439:Sulfhydryl Reagents
D000577:Amides; D002264:Carboxylic Acids; D005971:Glutamates; D013439:Sulfhydryl Reagents; D018698:Glutamic Acid; D004024:Dicyclohexylcarbodiimide; D006180:Proton-Translocating ATPases