Na+ binding of V-type Na+-ATPase in Enterococcus hirae.

Rotation catalysis theory has been successfully applied to the molecular mechanism of the ATP synthase (F(0)F(1)-ATPase) and probably of the vacuolar ATPase. We investigated the ion binding step to Enterococcus hirae Na(+)-translocating V-ATPase. The kinetics of Na(+) binding to purified V-ATPase suggested 6 +/- 1 Na(+) bound/enzyme molecule, with a single high affinity (K(d(Na(+()))) = 15 +/- 5 micrometer). The number of cation binding sites is consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. Release of the bound (22)Na(+) from purified molecules in a chasing experiment showed two phases: a fast component (about two-thirds of the total amount of bound Na(+); k(exchange) > 1.7 min(-1)) and a slow component (about one-third of the total; k(exchange) = 0.16 min(-1)), which changes to the fast component by adding ATP or ATPgammaS. This suggested that about two-thirds of the Na(+) binding sites of the Na(+)-ATPase are readily accessible from the aqueous phase and that the slow component is important for the transport reaction.