Abstract
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.
Highlights
Ion-transporting ATPases are divided into three types: PATPase, F-ATPase, and V-ATPase
This decrease of the rate was due to product inhibition by ADP, because ATPase activity in the presence of 1 mM ATP is strongly inhibited by 4 mM ADP. These findings suggest that the affinity of Naϩ-binding sites of the enzyme is lowered in the enzyme reaction
We demonstrated that affinity of hydrolysis because ATP analogs (AMP-PNP or ATP␥s) or aluminum fluoride, which is postulated to trap ATPases at their transition state, did not inhibit the Naϩ binding capacity significantly; 12% inhibition of Naϩ binding by fluoroaluminate may correspond to a decreased affinity of one of six Naϩbinding sites at the most, but the value is difficult to distinguish from the experimental error
Summary
Ion-transporting ATPases are divided into three types: PATPase, F-ATPase, and V-ATPase. V-ATPase is a proton pump in acidic organelles, plasma membranes of eukaryotic cells [4], and bacteria [5]. The reaction mechanism of P-type ATPase has been studied and explained by affinity change of the ATPase; ions are bound tightly on the low concentration side of the membrane and a conformational change caused by high energy intermediate of ATP hydrolysis results in their exposure to the high concentration side. The kinetics of Naϩ binding to purified V-ATPase suggested that 6 Ϯ 1 Naϩ ions bind per enzyme molecule with a single high affinity (Kd ϭ 15 Ϯ 5 M) [20]. Taking advantage of Naϩ binding by the V-ATPase, we further characterized the enzyme properties aiming to understand the above inconsistency and the ion-translocating mechanism of F- and V-type ATPases
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