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 P(i) was added along with ADp + Mg2+ or when ATP + Mg2+ was added to generate ADP + P(i) (+Mg2+) in the catalytic site. Trypsin treatment of ECF1F0 was also shown to increase enzymic activity on a time scale corresponding to that of the cleavage of the E subunit, indicating that the epsilon-subunit inhibits ATPase activity in ECF1F0. The ligand-dependent conformational changes in the epsilon-subunit were also examined in cross-linking experiments using the water-soluble carbodiimide I-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC). In the presence of ATP + Mg2+ or ADP + P(i) + Mg2+, the epsilon-subunit cross-linked to beta in high yield. With ATP + EDTA, the yield of beta-epsilon-cross-linked product was much reduced. Prior reaction of ECF1F0 with dicyclohexylcarbodiimide (DCCD), under conditions in which only the F(o) 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. We suggest that conformational rearrangements of the epsilon-subunit are a part of the coupling between events at the catalytic sites and proton translocation in the ATP synthase.