KINETICS OF ATP HYDROLYSIS BY THE F1-ATPASE FROM BACILLUS PS3 - A REAPPRAISAL OF THE EFFECTS OF ATP AND MG2+

ATPase activity of the F-1-ATPase from the thermophilic Bacillus PS3 ( TF1) was measured as a function of ATP concentration at thrice differe nt magnesium ion concentrations. A high-performance chromatographic me thod was used to determine directly ADP concentration in the reaction medium and to measure the steady-state rate of its appearance. Multiph asic curves of ATPase activity versus ATP concentration were obtained, with a first saturating rate mode at low ATP concentrations, a higher rate mode which became predominant at ATP concentrations depending on magnesium concentration, and a marked inhibition of ATP hydrolysis at high ATP concentrations. These curves could be simulated with equival ent residual enter either by assuming that the ATP-magnesium chelate i s the substrate of the enzyme, free magnesium being an inhibitor, or t hat free ATP is the substrate, free magnesium being an essential activ ator. In both cases, the observed hydrolysis rate was assumed to be th e sum of two independent rates with different kinetic parameters, as w ould be the case for an enzyme with functionally heterogeneous and ind ependent catalytic sites. Cross-checking of the different series of ki netic parameters with the binding affinity of TF1 for ATP, measured by a high-performance chromatographic method, is in favour of a model in which the hydrolysis rate is determined by the concentration of free ATP, free magnesium being an essential activator.