Inhibition of pyrophosphatase activity of mouse duodenal alkaline phosphatase by magnesium ions

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Abstract
Duodenal alkaline phosphatase of juvenile (11-day-old) mice, like other non-specific alkaline phosphatases, has the ability to hydrolyse PPi. When a constant Mg2+/PPi concentration ratio is maintained, plots of velocity as a function of PPi concentration are consistent with Michaelis–Menten kinetics. Mg2+ activates pyrophosphate hydrolysis and maximal activity is obtained at a constant Mg2+/PPi concentration ratio of 0·66. At higher ratios there is strong inhibition. At constant concentrations of Mg2+ and increasing concentrations of PPi, the velocity–substrate (PPi) concentration plots show sigmoidal dependence. By assuming that the true substrate is MgP2O72− complex, and using complexity constants, the concentrations of free Mg2+, Mg2P2O7 and MgP2O72− were calculated in assay mixtures ranging in PPi concentration from 0·1 to 2·5mm and in total Mg2+ concentration from 0·6 to 2·6mm. From these data, the concentrations of added Mg2+ and PPi in the assay mixtures were selected so that the velocity could be measured (1) at three fixed concentrations of free Mg2+ ions with varied concentrations of MgP2O72− and (2) at four fixed concentrations of Mg2P2O7 with varied concentrations of MgP2O72−. Lineweaver–Burk and Hill plots from these data showed that the inhibition is caused by free Mg2+ ions, of a mixed type and consistent with Michaelis–Menten kinetics. The sigmoidal dependence observed between velocity and PPi concentration at constant concentration of total Mg2+ is therefore not due to allosteric inhibition. It is due to a combined effect of (1) inhibition by free Mg2+ ions, (2) depletion of the true substrate, MgP2O72−, owing to the formation of Mg2P2O7 and (3) the manner in which the concentrations of these three molecular or ionic species change when PPi concentration is increased maintaining the total Mg2+ concentration constant.