Characterization of the membrane-bound inorganic pyrophosphatase in Rhodospirillum rubrum.

The membrane-bound inorganic pyrophosphatase (EC 3.6.1.1) from Rhodospirillum rubrum has been investigated with the tools of enzyme kinetics, and with two amino acid reagents, N-ethyl-maleimide (MalNET) and 4-chloro-7-nitrobenzofurazan (Nbf-Cl). 1. The concentration of the true substrate, MgPPi, was varied with constant concentrations of free Mg2+ or PPi. It was observed that Mg2+ acted as an activator. 2. Heat inactivation of the enzyme at 62 degrees C was slowed down in the presence of Mg2+. 3. MalNET and Nbf-Cl bind to the enzyme, and inhibit its activity. The effect of both reagents is dependent on the temperature. 4. A model is proposed where the 1:1 complex of Mg2+:PPi acts as substrate and Mg2+ interacts directly with the enzyme as an activator. PPi can bind to the enzyme, but is not hydrolyzed in the uncomplexed form.