ATP-dependent affinity change of Na+-binding sites of V-ATPase.

V-type Na(+)-ATPase of Enterococcus hirae binds about six (6 +/- 1) Na(+) ions/enzyme molecule with a high affinity (Murata, T., Igarashi, K., Kakinuma, Y., and Yamato, I. (2000) J. Biol. Chem. 275, 13415-13419). After the addition of 5 mm ATP, the binding capacity dropped to about 2 (1.8 +/- 0.3) Na(+) ions/enzyme molecule, returning to the initial value concomitant with the decrease of ATP hydrolysis rate. These findings suggest that the affinity of four of six Na(+)-binding sites of the enzyme changes (lowers) in enzyme reaction. The ATP analogs (adenosine 5'-O-(3-thiotriphosphate) or 5'-adenylylimido-diphosphate), ADP, or aluminum fluoride that is postulated to trap ATPases at their transition state did not inhibit the Na(+) binding capacity significantly. Therefore, the affinity decrease of Na(+)-binding sites was unlikely to be due to ATP binding alone or at the transition state of ATP hydrolysis. In the presence of 5 mm ATP, the ATPase showed strong negative cooperativity (n(H) = 0.16 +/- 0.03) for Na(+) stimulation of ATPase activity. The Hill coefficient (n(H)) increased to 1 in parallel to the decrease of ATP concentration in the reaction mixture. Thus, the ATP-dependent affinity change cooperatively occurs in continuous enzyme reaction.