The alpha 3(beta Y341W)3 gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 fails to dissociate ADP when MgATP is hydrolyzed at a single catalytic site and attains maximal velocity when three catalytic sites are saturated with MgATP.
The hydrolytic properties of the alpha3beta3gamma and mutant alpha3(betaY341W)3gamma subcomplexes of the TF1-ATPase have been compared. ATPase activity of the mutant is less sensitive to turnover-dependent inhibition by azide, less suppressed by increasing concentrations of Mg2+ during assay, and less stimulated by lauryl dimethylamine oxide (LDAO). Therefore, it has much lower propensity than wild-type to entrap inhibitory MgADP in a catalytic site during turnover. The fluorescence of the introduced tryptophans in the alpha3(betaY341W)3gamma subcomplex is completely quenched when catalytic sites are saturated with ATP or ADP with or without Mg2+ present. As reported for the betaY331W mutant of Escherichia coli F1 (Weber, J., Wilke-Mounts, S., Lee, R. S.-F., Grell, E., Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133), this provides a direct probe of nucleotide binding to catalytic sites. Addition of stoichiometric MgATP to the mutant subcomplex quenched one-third the tryptophan fluorescence which did not recover after 60 min. This was caused by entrapment of MgADP in a single catalytic site. Titration of catalytic sites of the alpha3(betaY341W)3gamma subcomplex with MgADP or MgATP revealed Kd's of < 50 nM, about 0.25 microM and about 35 microM. Titrations were not affected by azide, whereas LDAO lowered the affinities of catalytic sites 2 and 3 for MgADP by 5-fold and 2-fold, respectively. During titration with MgATP, LDAO slightly lowered affinity at ATP concentrations below 30 microM and had no effect at ATP concentrations above 30 microM. Maximal velocity was attained when the third catalytic site was titrated with MgATP in the presence or absence of LDAO. The same Kd's for binding MgATP to the (alphaA396C)3beta3(gammaA22C) mutant were observed before and after inactivating it by cross-linking alpha to gamma. This implies that the different affinities of catalytic sites for MgATP do not represent negative cooperativity, but rather represent heterogeneous affinities of catalytic sites dictated by the position of the coiled-coil of the gamma subunit within the central cavity of the (alpha beta)3 hexamer.
Dou
C
C
Department of Chemistry, University of California at San Diego, La Jolla 92093-0601, USA.
Fortes
P A
PA
Allison
W S
WS
D000244:Adenosine Diphosphate / Q000378:metabolism* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000494:pharmacology; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D002384:Catalysis / Q000187:drug effects; D002417:Cattle; D003432:Cross-Linking Reagents / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004123:Dimethylamines / Q000494:pharmacology; D005454:Fluorescence Polarization; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics*; D014443:Tyrosine / Q000235:genetics*
D001386:Azides; D003432:Cross-Linking Reagents; D003902:Detergents; D004123:Dimethylamines; D014443:Tyrosine; C014518:dodecyldimethylamine oxide; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases