New probes of the F-1-ATPase catalytic transition state reveal that two ofthe three catalytic sites can assume a transition state conformation simultaneously

MgADP in combination with fluoroscandium (ScFx) is shown to form a potently inhibitory, tightly bound, noncovalent complex at the catalytic sites of F -1-ATPase. The F1 . MgADP . ScFx complex mimics a catalytic transition stat e. Notably, ScFx caused large enhancement of MgADP binding affinity at both catalytic sites 1 and 2, with little effect at site 3. These results indic ate that sites 1 and 2 may form a transition state conformation. A new dire ct optical probe of F-1-ATPase catalytic transition state conformation is a lso reported, namely, substantial enhancement of fluorescence emission of r esidue beta-Trp-148 observed upon binding of MgADP . ScFx or MgIDP . ScFx. Using this fluorescence signal, titrations were performed with MgIDP . ScFx which demonstrated that catalytic sites 1 and 2 can both form a transition state conformation but site 3 cannot. Supporting data were obtained using MgIDP-fluoroaluminate. Current models of the MgATP hydrolysis mechanism uni formly make the assumption that only one catalytic site hydrolyzes MgATP at any one time. The fluorometal analogues demonstrate that two sites have th e capability to form the transition state simultaneously.