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 c onformational change in the gamma subunit. This change can be monitore d by fluorescence changes in 7-(diethylamino)-4-methyl]coumarin-3-yl)] maleimide (CM) bound at a cysteine introduced by site-directed mutagen esis 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 us ing the slowly cleavable ATP gamma S. The data indicate that maximal f luorescence change occurs with binding of 1 mol of nucleotide triphosp hate per mole of ECF(1). During unisite catalysis, ATP binding causes a fluorescence enhancement from CM bound at position 106, which is the n followed by fluorescence quenching. The kinetics of these fluorescen ce changes have been measured using both ATP and ATP gamma S as substr ate. With ATP gamma S, these kinetics can be simulated using rate cons tants similar to those for ATP except for an approximately 30-fold slo wer 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 whi ch the bond cleavage or the P-i release step was responsible for fluor escence quenching. The results indicate that ATP or ATP gamma S bindin g 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.