ATP Binding Causes a Conformational Change in the γ Subunit of the Escherichia coli F1ATPase Which Is Reversed on Bond Cleavage

ATP hydrolysis by the Escherichia coli F1 ATPase (ECF1) induces a conformational change in the 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 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 ATPS.The data indicate that maximal fluorescence change occurs with binding of 1 mol of nucleotide triphosphate per mole of ECF1. 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 ATPS as substrate. With ATPS, 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 ATPS were analyzed by simulations in which the bond cleavage or the Pi release step was responsible for fluorescence quenching. The results indicate that ATP or ATPS binding causes the fluorescence enhancement of CM bound to the γ subunit and that this conformational change is reversed upon bond cleavage to yield ADP•Pi or ADP•PiS in catalytic sites. © 1994, American Chemical Society. All rights reserved.