MUTATIONS IN THE NUCLEOTIDE-BINDING DOMAIN OF THE ALPHA-SUBUNITS OF THE F1-ATPASE FROM THERMOPHILIC BACILLUS PS3 THAT AFFECT CROSS-TALK BETWEEN NUCLEOTIDE-BINDING SITES

Inactivation of MF1 (bovine mitochondrial F-1-ATPase) with 5'-p-fluoro sulfonylbenzoylethenoadenosine is caused by labeling alpha Y244 [Verbu rg, J. G., and Allison, W. S. (1990) J. Biol. Chem. 265, 8065-8074]. I n the crystal structure [Abrahams, J. P., Leslie, A. G, W., Lutter, R. , and Walker, J. E, (1994) Nature 370, 621-628], alpha Y244 is hydroge n bonded to alpha R304 which is also hydrogen bonded to alpha Y300, Th e catalytic properties of mutant alpha(3) beta(3) gamma subcomplexes o f the TF1-ATPase from the thermophilic Bacillus PS3 containing the alp ha F244C, alpha R304C, and alpha Y300C substitutions have been examine d. Each has unique features for hydrolyzing ATP and forming inhibitory ADP-fluoroaluminate complexes in catalytic sites. Unlike wild-type, t he (alpha R304C)(3) beta(3) gamma and (alpha Y300C)(3) beta(3) gamma s ubcomplexes entrap inhibitory MgADP in a catalytic site during turnove r which fails to dissociate when ATP binds to noncatalytic sites. Alth ough the hydrolytic properties of the (alpha F244C)(3) beta(3) gamma s ubcomplex and wild-type are similar, the mutant forms ADP-fluoroalumin ate complexes 7 times faster than wild-type when Al3+ and F-are added to it in the presence of excess ADP and Mg2+. It also resists inhibiti on by high Mg2+ concentrations in the assay medium. At least one nonca talytic site of the (alpha F244C)(3) beta(3) gamma subcomplex has incr eased affinity for ADP, indicating that the enhanced rate of formation of the ADP-fluoroaluminate complex reflects augmented cooperativity b etween noncatalytic and catalytic sites. The rate of formation of the ADP-fluoroaluminate complex in (alpha Y300C)(3) beta(3) gamma increase s only 40% when MgADP in bound to two catalytic sites rather than one, compared to a 9-fold increase exhibited by wild type. When Al3+ and F - are added to the (alpha Y300C)(3) beta(3) gamma subcomplex after inc ubation with excess ADP and Mg2+, ADP-fluoroaluminate complexes are fo rmed in three catalytic sites rather than two observed with the other subcomplexes, Reconciliation of the catalytic properties of the mutant subcomplexes in terms of the crystal structure suggests that alpha F2 44, alpha R304, and alpha Y300 of TF1 are part of a pathway that propa gates conformational signals from one catalytic site to another.