Catalytic activity of the alpha3beta3gamma complex of F1-ATPase without noncatalytic nucleotide binding site.
A mutant alpha3beta3gamma complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 microM; Km(2), 135 microM) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1). In the presence of ATP, the wild-type complex dissociated the inhibitory [3H]ADP preloaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [3H]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below approximately 300 microM.
Matsui
T
T
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan.
Muneyuki
E
E
Honda
M
M
Allison
W S
WS
Dou
C
C
Yoshida
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001407:Bacillus; D001665:Binding Sites; D004123:Dimethylamines / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D010449:Peptide Mapping; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013501:Surface-Active Agents / Q000378:metabolism
D004123:Dimethylamines; D005456:Fluorescent Dyes; D013501:Surface-Active Agents; C014518:dodecyldimethylamine oxide; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases