The activity of CF1-ATPase was inhibited by vanadate in an allosteric manner with respect to CaATP as substrate. The cooperative interaction was enhanced by preincubation of the enzyme in the presence of ADP and Ca2+ ions and of free divalent metal ions during assay of the activity. The strongest cooperative interaction with a Hill coefficient of 5.3 ± 0.1 was found when the reaction was stopped after 30 s, before steady state was reached. Under these conditions, the concentration of an exchangeable ADP, tightly bound to one of the active sites on the enzyme, was shown to be the highest. A Ki of 12.4 ± 1.2 μM for vanadate inhibition was determined under these conditions. Direct measurements with the aid of 51V NMR indicated that vanadate binds to CF1 in the presence of Ca2+ and ADP in a positive cooperative manner with a Hill coefficient of 2.3 ± 0.2 and an average Kd of 0.3 ± 0.04 nM. It was suggested that a formation of pentacovalent vanadyl-ADP at the active site caused the inhibition. Vanadyl-ADP was suggested to be a strong inhibitor, being an analogue of a pentacovalent phosphoryl-ADP, which is proposed to be the transition state intermediate of CF1.
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 15 Jun 1993|