Nucleotide-dependent and dicyclohexylcarbodiimide-sensitive conformational changes in the epsilon subunit of Escherichia coli ATP synthase.
The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1F0 (ECF1F0) is shown to be ligand-dependent as measured by Western analysis using monoclonal antibodies. The cleavage of the epsilon subunit was rapid in the presence of ADP alone, ATP + EDTA, or AMP-PNP + Mg2+, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site. Trypsin treatment of ECF1Fo was also shown to increase enzymic activity on a time scale corresponding to that of the cleavage of the epsilon subunit, indicating that the epsilon subunit inhibits ATPase activity in ECF1Fo. The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC). In the presence of ATP + Mg2+ or ADP + Pi + Mg2+, the epsilon subunit cross-linked product was much reduced. Prior reaction of ECF1Fo with dicyclohexylcarbodiimide (DCCD), under conditions in which only the Fo part was modified, blocked the conformational changes induced by ligand binding. When the enzyme complex was reacted with DCCD in ATP + EDTA, the cleavage of the epsilon subunit was rapid and yield of cross-linking of beta to epsilon subunit low, whether trypsin cleavage was conducted in ATP + EDTA or ATP + Mg2+. When enzyme was reacted with DCCD in ATP + Mg2+, cleavage of the epsilon subunit was slow and yield of cross-linking of beta to epsilon high, under all nucleotide conditions for proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Mendel-Hartvig
J
J
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Capaldi
R A
RA
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D046911:Macromolecular Substances; D004533:Egtazic Acid; D004024:Dicyclohexylcarbodiimide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases; D005022:Ethyldimethylaminopropyl Carbodiimide