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

The causes of inhibition of Escherichia coli inorganic pyrophosphatase (PPa se) by Ca2+ were investigated. The interactions of several mutant pyrophosp hatases with Ca2+ in the absence of substrate were analyzed by equilibrium dialysis. The kinetics of Ca2+ inhibition of hydrolysis of the substrates M gPPi and LaPPi by the native PPase and three mutant enzymes (Asp-42-Asn, Al a, and Glu) were studied. X-Ray data on E. coli PPase complexed with Ca2+ o r CaPPi solved at atomic resolution were analyzed. It was shown that, in th e course of the catalytic reaction, Ca2+ replaces Mg2+ at the M2 site, whic h shows higher affinity for Ca2+ than for Mg2+. Different properties of the se cations account for active site deformation. Our findings indicate that the filling of the M2 site with Ca2+ is sufficient for PPase inhibition. Th is fact proves that Ca2+ is incapable of properly activating the H2O molecu le 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 ail known PPases. Other possible reasons for the inhibitory effect of Ca2on the enzyme activity are also considered.