In vivo affinity label of a protein expressed in Escherichia coli. Coenzyme A occupied the AT(D)P binding site of the mutant F1-ATPase beta subunit (Y307C) through a disulfide bond.
When Tyr-307 of the beta subunit of F1-ATPase from a thermophilic Bacillus strain PS3 is replaced by cysteine and expressed in Escherichia coli cells, about a half population of the mutant beta subunit are labeled by Coenzyme A at Cys-307 through a disulfide bond which is cleavable by reducing treatment. The mutant beta subunit can be reconstituted into the alpha 3 beta 3 complex of which ATPase activity is stimulated two-fold by reducing treatment either prior or after reconstitution. Since Tyr-307 has been supposed to be located at one of subdomains which form the ATP binding site of the beta subunit, Coenzyme A binds to the mutant beta subunit as an AT(D)P analogue in E. coli cells and then covalently attaches to Cys-307.
Odaka
M
M
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Yokohama, Japan.
Kiribuchi
K
K
Allison
W S
WS
Yoshida
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels; D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli; D009154:Mutation; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D000345:Affinity Labels; D001426:Bacterial Proteins; D004220:Disulfides; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D003545:Cysteine; D003065:Coenzyme A