Reversible inhibition of Escherichia coli inorganic pyrophosphatase by fluoride: trapped catalytic intermediates in cryo-crystallographic studies.

Here, we describe high-resolution X-ray structures of Escherichia coli inorganic pyrophosphatase (E-PPase) complexed with the substrate, magnesium, or manganese pyrophosphate. The structures correspond to steps in the catalytic synthesis of enzyme-bound pyrophosphate (PP(i)) in the presence of fluoride as an inhibitor of hydrolysis. The catalytic reaction intermediates were trapped applying a new method that we developed for initiating hydrolytic activity in the E-PPase crystal. X-ray structures were obtained for three consecutive states of the enzyme in the course of hydrolysis. Comparative analysis of these structures showed that the Mn2+-supported hydrolysis of the phosphoanhydride bond is followed by a fast release of the leaving phosphate from the P1 site. The electrophilic phosphate P2 is trapped in the "down" conformation. Its movement into the "up" position most likely represents the rate-limiting step of Mn2+-supported hydrolysis. We further determined the crystal structure of the Arg43Gln mutant variant of E-PPase complexed with one phosphate and four Mn ions.

Effect of mutation of the conservative glycine residues Gly100 and Gly147 on stability of Escherichia coli inorganic pyrophosphatase.

Sequence alignment of inorganic pyrophosphatases (PPases) isolated from the different organisms shows that glycine residues Gly100 and Gly147 are conservative. These residues are located in flexible segments of a polypeptide chain that have similar structure in the different PPases. To elucidate the possible role of these segments in the functioning of PPase, the mutant variants Gly100Ala and Gly147Val in conservative loops have been obtained. In this work, the influence of these mutations on stability of PPase globular structure has been studied. Differential scanning calorimetry has been used to determine the apparent enthalpy of thermal denaturation for the native PPase and its mutant variants Gly100Ala and Gly147Val. Guanidine hydrochloride-induced chemical denaturation of PPase has also been studied. It is shown that the substitutions of Gly100 and Gly147 result in overall destabilization of the globular structure.

Substitutions of glycine residues Gly100 and Gly147 in conservative loops decrease rates of conformational rearrangements of Escherichia coli inorganic pyrophosphatase.

Escherichia coli inorganic pyrophosphatase (PPase) is a one-domain globular enzyme characterized by its ability to easily undergo minor structure rearrangements involving flexible segments of the polypeptide chain. To elucidate a possible role of these segments in catalysis, catalytic properties of mutant variants of E. coli PPase Gly100Ala and Gly147Val with substitutions in the conservative loops II and III have been studied. The main result of the mutations was a sharp decrease in the rates of conformational changes required for binding of activating Mg2+ ions, whereas affinity of the enzyme for Mg2+ was not affected. The pH-independent parameters of MgPP(i) hydrolysis, kcat and kcat/Km, have been determined for the mutant PPases. The values of kcat for Gly100Ala and Gly147Val variants were 4 and 25%, respectively, of the value for the native enzyme. Parameter kcat/Km for both mutants was two orders of magnitude lower. Mutation Gly147Val increased pH-independent Km value about tenfold. The study of synthesis of pyrophosphate in the active sites of the mutant PPases has shown that the maximal level of synthesized pyrophosphate was in the case of Gly100Ala twofold, and in the case of Gly147Val fivefold, higher than for the native enzyme. The results reported in this paper demonstrate that the flexibility of the loops where the residues Gly100 and Gly147 are located is necessary at the stages of substrate binding and product release. In the case of Gly100Ala PPase, significant impairment of affinity of enzyme effector site for PP(i) was also found.