Do ATP 4− and Mg 2+ bind stepwise to the F 1 ‐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 Mg 2+ 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 Mg 2+ to the E ATP 4− complex is the key reaction towards the formation of the ternary complex. These models (and 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 [Mg 2+ ]. The rates were asymmetrical with respect to increasing [ATP 4− ] versus increasing [Mg 2+ ]. For the ordered‐binding alternative, a series of apparent dissociation constants were obtained for ATP 4− (.cf2.K.cf2..cf1..esapp.rb.eiA.rb), which decreased as [Mg 2+ ] increased. From this family of .cf2.K.cf2..cf1..esapp.rb.eiA.rb the true K A was retrieved by extrapolation to [Mg 2+ ] = 0 and was found to be 0.2 mM. The dissociation constants for Mg 2+ , established from these experiments, were also apparent (.cf2.K.cf2..cf1..esapp.rb.eiB.rb) and dependent on [ATP 4− ] as well as on the pH. The actual K B was established from a series of .cf2.K.cf2..cf1..esapp.rb.eiB.rb by extrapolating to [ATP 4− ] = ∞ and to the absence of competing protons, and was found to be 0.0041 mM. The p K a of the protonable group for Mg 2+ 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 Mg 2+ cannot bind once the E ATP 4− complex is formed, while in the ordered‐binding model binding of Mg 2+ 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.