Inhibition and inactivation of the F1 adenosinetriphosphatase from Bacillus PS3 by dequalinium and activation of the enzyme by lauryl dimethylamine oxide.
The F1-ATPase from Bacillus PS3 (TF1) hydrolyzes 50 microM ATP in three kinetic phases. An initial burst rapidly decelerates to a partially inhibited, intermediate phase, which, in turn, 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 Km values of 770 microM and 144 microM, respectively, when obtained in the absence of LDAO. The apparent Km values determined from the data obtained in the presence of LDAO for the same phases are 303 microM and 163 microM, 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 Kd of 12.5 microM in the presence or absence of LDAO.(ABSTRACT TRUNCATED AT 250 WORDS)
Paik
S R
SR
Department of Chemistry, University of California at San Diego, La Jolla 92093-0601.
Jault
J M
JM
Allison
W S
WS
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002250:Carbon Radioisotopes; D003488:Cyanogen Bromide; D003868:Dequalinium / Q000378:metabolism / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology; D004123:Dimethylamines / Q000494:pharmacology*; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014357:Trypsin
D002250:Carbon Radioisotopes; D003902:Detergents; D004123:Dimethylamines; D010446:Peptide Fragments; C014518:dodecyldimethylamine oxide; D003868:Dequalinium; D014357:Trypsin; D006180:Proton-Translocating ATPases; D003488:Cyanogen Bromide