The role of Asp42 in Escherichia coli inorganic pyrophosphatase functioning

Excess of Mg2+ ions is known to inhibit the soluble inorganic pyrophosphata ses (PPases). In contrast, the mutant Escherichia coli inorganic pyrophosph atase Asp42-->Asn is three times more active than native and retains its ac tivity at high Mg2+ concentration. In this paper, another two mutant varian ts with Asp42 replaced by Ala or Glu were investigated to characterize the role of Asp42 in catalysis. pH-independent kinetic parameters of MgPPi hydr olysis and the dissociation constants for the activating and inhibitory Mg2 + ions were calculated. It was shown that Mg2+ inhibition of MgPPi hydrolys is by native PPase exhibited uncompetitive kinetics under the saturating su bstrate concentration. All three substitutions of Asp42 lead to a sharp dec rease of inhibitory Mg2+ affinity to the enzyme. These findings allow deter mination of the sites of inhibitory and substrate Mg2+ ions binding to PPas e. Common features of these mutants allow the conclusion that the function of Asp42 is to accurately coordinate the residues implicated in the substra te and the inhibitory Mg2+ ion binding to PPase active site. Structural ana lysis of PPase complexed with Mg2+ compared with PPase complexed with Mn2and reaction products confirms this supposition.