CATALYTIC SITE NUCLEOTIDE AND INORGANIC-PHOSPHATE DEPENDENCE OF THE CONFORMATION OF THE EPSILON-SUBUNIT IN ESCHERICHIA-COLI ADENOSINE-TRIPHOSPHATASE

The rate of trypsin cleavage of the epsilon-subunit of Escherichia coli F1 (ECF1) has been found to be ligand-dependent, as measured indirectly by the activation of the enzyme that occurs on protease digestion, or when followed directly by monitoring the cleavage of this subunit using monoclonal antibodies. The cleavage of the epsilon-subunit was fast in the presence of ADP alone, ADP + Mg2+, ATP + EDTA, or AMP-PNP, 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(s). The half-maximal concentration of P(i) required in the presence of ADP + Mg2+ to protect the epsilon-subunit from cleavage by trypsin was 50-mu-M, which is in the range measured for the high-affinity binding of P(i) to F1. 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 + Mg2+ + P(i), the epsilon-subunit cross-linked to beta in high yield. With ATP + EDTA or ADP + Mg2+ (no P(i)), the yield of the beta-epsilon cross-linked product was much reduced. We conclude that the epsilon-subunit undergoes a conformational change dependent on the presence of P(i). It has been found previously that binding of the epsilon-subunit to ECF1 inhibits ATPase activity by decreasing the off rate of P(i) [Dunn, S. D., Zadorozny, V. D., Tozer R. G., & Orr, L. E. (1987) Biochemistry 26, 4488-4493]. This reciprocal relationship between P(i) binding and epsilon-subunit conformation has important implications for energy transduction by the E. coli ATP synthase.