ATP BINDING CAUSES A CONFORMATIONAL CHANGE IN THE GAMMA-SUBUNIT OF THE ESCHERICHIA-COLI F(1)ATPASE WHICH IS REVERSED ON BOND-CLEAVAGE

ATP hydrolysis by the Escherichia coli F-1 ATPase (ECF(1)) induces a conformational change in the gamma subunit. This change can be monitored by fluorescence changes in N-[4-[7-(diethylamino)-4-methyl]coumarin-3-yl)]maleimide (CM) bound at a cysteine introduced by site-directed mutagenesis into the gamma subunit at position 106 [Turina, P., & Capaldi, R. A. (1994) J. Biol. Chem. 269, 13465-13471]. In studies reported here, the magnitude of the fluorescence change has been determined with the noncleavable nucleotide analogue AMP.PNP and by rapid measurements using the slowly cleavable ATP gamma S. The data indicate that maximal fluorescence change occurs with binding of 1 mol of nucleotide triphosphate per mole of ECF(1). During unisite catalysis, ATP binding causes a fluorescence enhancement from CM bound at position 106, which is then followed by fluorescence quenching. The kinetics of these fluorescence changes have been measured using both ATP and ATP gamma S as substrate. With ATP gamma S, these kinetics can be simulated using rate constants similar to those for ATP except for an approximately 30-fold slower rate of the bond cleavage and resynthesis steps, i.e., k(+2) and k(-2). The observed rates and amplitudes of the fluorescence changes on hydrolysis of ATP and ATP gamma S were analyzed by simulations in which the bond cleavage or the P-i release step was responsible for fluorescence quenching. The results indicate that ATP or ATP gamma S binding causes the fluorescence enhancement of CM bound to the gamma subunit and that this conformational change is reversed upon bond cleavage to yield ADP.P-i or ADP.PiS in catalytic sites.