The characteristics of the (αV371C)3(βR337C)3γ double mutant subcomplex of the TF1-ATPase indicate that the catalytic site at the αTP-βTP interface with bound MgADP in crystal structures of MF1 represents a catalytic site containing inhibitory MgADP

In the MF1 crystal structure with the MgADP-fluoroaluminate complex bound to two catalytic sites [Menz, R. I., Walker, J. E., and Leslie, A. G. W. (2001) Cell 106, 331-341], the guanidinium of betaR(337) is within 2.9 Angstrom of the alpha-oxygen of alphaS(370) and 3.7 Angstrom of a methyl group of alphaV(371) at the alphaE-beta(HC) interface. To examine the functional role of this contact, the (alphaV(371)C)(3)(betaR(337)C)(3)gamma subcomplex of the TF1-ATPase was prepared and characterized. Steady state ATPase activity of the reduced double-mutant is 30% of that of the wild type. Inactivation of the double mutant containing empty catalytic sites or MgADP bound to one catalytic site with CuCl2 cross-linked two alpha-beta pairs, whereas a single alpha-beta pair cross-linked when at least two catalytic sites contained MgADP. The reduced double mutant hydrolyzed substoichiometric ATP 100-fold more rapidly than the enzyme containing two cross-linked alpha-beta pairs. Addition of AlCl3 and NaF to the reduced double mutant after incubation with stoichiometric MgADP or 200 muM MgADP irreversibly inactivated the steady state ATPase activity with rate constants of 1.5 x 10(-2) and 4.1 x 10(-2) min(-1), respectively. In contrast, addition of AlCl3 and NaF to the cross-linked enzyme after incubation with stoichiometric or 200 muM MgADP irreversibly inactivated ATPase activity with a common rate constant of similar to10(-4) min(-1). Correlation of these results with crystal structures of MF1 suggests that the catalytic site at the alpha(TP)-beta(TP) interface is loaded first upon addition of nucleotides to nucleoticle-depleted F-1-ATPases and that the catalytic site at the alpha(TP)-beta(TP) interface with bound MgADP in crystal structures represents a catalytic site containing inhibitory MgADP.