Derivatization of the catalytic subunits of the chloroplast ATPase by 2-azido-ATP and dicyclohexylcarbodiimide. Evidence for catalytically induced interchange of the subunits.
Modifications of the catalytic beta subunits of the chloroplast ATPase (CF1-ATPase) are reported which support the proposal that all three subunits participate sequentially during catalysis. The beta subunits of the CF1-ATPase are sufficiently homogeneous to allow detection of their derivatization with dicyclohexylcarbodiimide (DCCD) or the substrate analog 2-azido-ATP by two-dimensional isoelectric focusing. Whether the DCCD reacts with the same beta subunit that tightly binds ATP has not been known. Our results show that when CF1-ATPase is covalently labeled with 2-azido-ATP followed by reaction with DCCD, different beta subunits are labeled. The DCCD labeling does not stop catalytic cooperativity of the CF1-ATPase and allows slow enzyme turnover. When the DCCD-modified enzyme catalyzes 2-azido-ATP cleavage and the enzyme with tightly bound nucleotide is photolyzed, both DCCD-modified and unmodified subunits are randomly labeled by the azido nucleotide. This result is as expected if during the catalytic cycle one beta subunit with unique properties is replaced by another subunit that gains these properties. The participation of all three subunits in the catalytic cycle is suggested by the apparent retention of catalytic cooperativity by the two remaining subunits after one subunit has already catalyzed 2-azido-ATP cleavage and been labeled.
Melese
T
T
Boyer
P D
PD
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology*; D002234:Carbodiimides / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D001386:Azides; D002234:Carbodiimides; D046911:Macromolecular Substances; D004024:Dicyclohexylcarbodiimide; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases