INHIBITION AND INACTIVATION OF THE F(1)-ADENOSINE-TRIPHOSPHATASE FROM BACILLUS PS3 BY DEQUALINIUM AND ACTIVATION OF THE ENZYME BY LAURYL DIMETHYLAMINE OXIDE

The F1-ATPase from Bacillus PS3 (TF1) hydrolyzes 50 muM ATP in three kinetic phases. An initial burst rapidly decelerates to a partially inhibited, intermediate phase, which, in tum, gradually accelerates to an uninhibited, final steady-state rate. Lauryl dimethylamine oxide (LDAO) stimulates the final rate over 4-fold. The stimulatory effect saturates at about 0.1% LDAO. Under these conditions, the intermediate phase is nearly absent. Dequalinium inhibits TF1 reversibly in the dark in the presence or absence of LDAO. The apparent affinity of TF1 for dequalinium increases in the presence of LDAO. Dixon plots of the initial rates of the intermediate phase and the final rates against dequalinium concentration at a series of fixed ATP concentrations in the presence and absence of 0.03% LDAO indicate noncompetitive inhibition in each case. Replots of the slopes of the Dixon plots for the initial rate of the intermediate phase and the final rate against 1/[ATP] reveal apparent K(m) values of 770 muM and 144 muM, respectively, when obtained in the absence of LDAO. The apparent K(m) values determined from the data obtained in the presence of LDAO for the same phases are 303 muM and 163 muM, respectively. These results suggest that LDAO stimulates ATPase activity either by increasing the affinity of noncatalytic sites for ATP, which promotes release of inhibitory MgADP from a catalytic site, or by directly promoting release of MgADP from the affected catalytic site. Dequalinium retards this process without affecting the affinity of noncatalytic sites for ATP. When irradiated in the presence of dequalinium, TF1 is rapidly inactivated with an apparent K(d) of 12.5 AM in the presence or absence of LDAO. Under both conditions, 90% photoinactivation occurs with incorporation of about 2-2.5 mol of [C-14]dequalinium/mol of TF1. Fractionation of cyanogen bromide-tryptic digests of enzyme photoinactivated with [C-14]dequalinium with or without LDAO led to isolation of a labeled peptide containing residues 406-431 of the beta subunit in which Phe-420 is derivatized. The results obtained indicate that photoinactivation of TF1 with dequalinium is accompanied by derivatization of Phe-420 in a single copy of the beta subunit and that the residual radioactivity incorporated is the consequence of nonspecific labeling.