Mechanism of Ca2+-induced inhibition of Escherichia coli inorganic pyrophosphatase.

The causes of inhibition of Escherichia coli inorganic pyrophosphatase (PPase) by Ca2+ were investigated. The interactions of several mutant pyrophosphatases with Ca2+ in the absence of substrate were analyzed by equilibrium dialysis. The kinetics of Ca2+ inhibition of hydrolysis of the substrates MgPPi and LaPPi by the native PPase and three mutant enzymes (Asp-42-Asn, Ala, and Glu) were studied. X-Ray data on E. coli PPase complexed with Ca2+ or CaPPi solved at atomic resolution were analyzed. It was shown that, in the course of the catalytic reaction, Ca2+ replaces Mg2+ at the M2 site, which shows higher affinity for Ca2+ than for Mg2+. Different properties of these cations account for active site deformation. Our findings indicate that the filling of the M2 site with Ca2+ is sufficient for PPase inhibition. This fact proves that Ca2+ is incapable of properly activating the H2O molecule for nucleophilic attack on PPi. It was also demonstrated that Ca2+, as a constituent of the non-hydrolyzable substrate analog CaPPi, competes with MgPPi at the M3 binding site. As a result, Ca2+ is a powerful inhibitor of all known PPases. Other possible reasons for the inhibitory effect of Ca2+ on the enzyme activity are also considered.