Ichiro Yamato

ATP-dependent Affinity Change of Na+-binding Sites of V-ATPase

V-type Na+-ATPase ofEnterococcus 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.