Rapid hydrolysis of ATP by mitochondrial F1-ATPase correlates with the filling of the second of three catalytic sites.

Strong positive catalytic cooperativity is a central feature of the binding change mechanism for F1-ATPases. However, a detail of the mechanism that remains controversial is whether the kinetic enhancement derived from using substrate-binding energy at one catalytic site to promote product release from another site occurs upon the filling of the second or third of three catalytic sites on F1. To address this question, we compare the ATP concentration dependence of the rate of ATP hydrolysis by F1 from beef heart mitochondria to the ATP concentration dependence of the level of occupancy of catalytic sites during steady-state catalysis as measured by a centrifuge filtration assay. A single Km(ATP) is observed at 77 +/- 6 microM. Analysis of the nucleotide-binding data shows that half-maximal occupancy of a second catalytic site occurs at 78 +/- 18 microM ATP. We conclude that ATP binding to a second catalytic site is sufficient to support rapid rates of catalysis.