Do ATP4- and Mg2+ bind stepwise to the F1-ATPase of Halobacterium saccharovorum?

It is commonly believed that MgATP(2-) is the substrate of F-1-ATPases and ATP(4-) acts as a competitive inhibitor. However, the velocity equation for such competitive inhibition is equivalent to that for a rapid equilibrium ordered binding mechanism in which ATP(4-) adds first and the binding of Mg2+ is dependent on the formation of the E.ATP(4-) complex. According to this ordered-binding model, solution formed MgATP(2-) is not recognized by the ATPase as a direct substrate, and the high-affinity binding of Mg2+ to the E.ATP(4-) complex is the key reaction towards the formation of the ternary complex. These models land others) were tested with an F-1-ATPase, isolated from Halobacterium saccharovorum, by evaluating the rate of ATP hydrolysis as a function of free [ATP(4-)] or free [Mg2+]. The rates were asymmetrical with respect to increasing [ATP(4-)] versus increasing [Mg2+]. For the ordered-binding alternative, a series of apparent dissociation constants were obtained for ATP(4-) (K-A(app)), which decreased as [Mg2+] increased. From this family of K-A(app) the true K-A was retrieved by extrapolation to [Mg2+] = 0 and was found to be 0.2 mM. The dissociation constants for Mg2+, established from these experiments, were also apparent (K-B(app)) and dependent on [ATP(4-)] as well as on the pH. The actual K-B was established from a series of K-B(app) by extrapolating to [ATP(4-)] = infinity and to the absence of competing protons, and was found to be 0.0041 mM. The pK(a) of the protonable group for Mg2+ binding is 8.2. For the competitive inhibition alternative, rearrangement of the constants and fitting to the velocity equation gave an actual binding constant for MgATP(2-) (K-EAB) of 0.0016 mM and for ATP(4-) (K-EA) of 0.2 mM. Decision between the two models has far-reaching mechanistic implications. In the competitive inhibition model MgATP(2-) binds with high affinity, but Mg2+ cannot bind once the E.ATP(4-) complex is formed, while in the ordered-binding model binding of Mg2+ requires that ATP(4-) adds first. The steric constraints evident in the diffraction structure of the ATP binding site in the bovine mitochondrial F-ATPase [Abrahams, J. P., Leslie, A. G. W., Lutter, R. & Walker, J. E. (1994) Nature 370, 621-628] tend to favor the ordered-binding model, but the final decision as to which kinetic model is valid has to be from further structural studies. If the ordered-binding model gains more experimental support, a revision of the current concepts of unisite catalysis' and negative cooperativity of nucleotide binding will be necessary.