The substrate for the hydrolytic activity of membrane-bound pyrophosphatase is the PP(i)-Mg2+ complex. The enzyme has no activity when the free Mg2+ concentration is lower than 10 ?M (at 0.5 mM-PP(i)-Mg2+), and therefore free Mg2+ is an essential activator of the hydrolytic activity. The Km for the substrate changes in response to variation in free Mg2+ concentration. from 10.25 to 0.6 mM when free Mg2+ is increased from 0.03 to 1.0 mM respectively. The K(m) for Mg2+ depends on the substrate concentration: the Km decreases from 0.52 to 0.14mM from 0.25 to 0.75mM-PP(i)-Mg2+ respectively. The extrapolated Km for Mg2+ in the absence of the substrate is 0.73 mM. Imidodiphosphate Mg2+ and free Ca2+ were used as competitive inhibitors of substrate and activator respectively. The equilibrium binding kinetics suggest an ordered mechanism for the activator and the substrate: Mg2+ ions bind the enzyme before PP(i)-Mg(2+) in the formation of the catalytic complex, membrane-bound pyrophosphatase-(Mg2+)-(PP(i)-Mg2+).
Última actualización: 26/09/2016