Kinetics of ATP hydrolysis by the F 1-ATPase from Bacillus PS3: a reappraisal of the effects of ATP and Mg 2+

Abstract

ATPase activity of the F 1-ATPase from the thermophilic Bacillus PS3 (TF 1) was measured as a function of ATP concentration at three different magnesium ion concentrations. A high-performance chromatographic method was used to determine directly ADP concentration in the reaction medium and to measure the steady-state rate of its appearance. Multiphasic 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 equivalent residual error either by assuming that the ATP-magnesium chelate is the substrate of the enzyme, free magnesium being an inhibitor, or that free ATP is the substrate, free magnesium being an essential activator. In both cases, the observed hydrolysis rate was assumed to be the sum of two independent rates with different kinetic parameters, as would be the case for an enzyme with functionally heterogeneous and independent catalytic sites. Cross-checking of the different series of kinetic parameters with the binding affinity of TF 1 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.