Binding of substrate at the effector site of pyrophosphatase increases the rate of its hydrolysis at the active site.

It is shown that in addition to the active site, each subunit of Escherichia coli inorganic pyrophosphatase (E-PPase) contains an extra binding site for the substrate magnesium pyrophosphate or its non-hydrolyzable analog magnesium methylenediphosphonate. The occupancy of the extra site stimulates the substrate conversion. Binding affinity of this site decreased or disappeared upon the conversion of E-PPase into a trimeric form or introduction of point mutations. However, when the slowly hydrolyzed substrate, lanthanum pyrophosphate (LaPP(i)), is used, the extra site was revealed in all enzyme forms of E-PPase and of Y-PPase (Saccharomyces cerevisiae PPase), resulting in about 100-fold activation of hydrolysis. A hypothesis on the localization of the extra site and the mechanism of its effect in E-PPase is presented.

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.

[A cationic cluster of amino acid residues of inorganic pyrophosphatase from Escherichia coli as a possible site of effector binding].

A computer-assisted analysis of the molecule of Escherichia coli pyrophosphatase was earlier used to localize the site capable of binding free pyrophosphate or methylene diphosphonate, a PPi analogue, and thereby activating the enzyme. A cluster of positively charged amino acid residues (Lys146, Lys148, Lys115, and Arg43) was revealed, and Lys115Ala, Lys148Gln, and Arg43Gln mutant pyrophosphatases (PPases) were obtained. It was shown that the kinetics of hydrolysis of the magnesium pyrophosphate (MgPPi) substrate by these mutant variants does not obey the Michaelis-Menten equation, which is expressed in two slopes in the double-reciprocal plot of the enzyme reaction rate vs. substrate concentration. The two regions on the curves correspond to the ranges of high and low MgPPi concentrations. This suggests that, in all mutant variants of the enzyme, the binding of PPi at the effector site becomes worse, whereas the affinity of MgPPi for the active site remains practically unchanged. Other properties of the enzymes, such as its oligomeric state, resistance to thermal denaturation, and resistance to the denaturing agent guanidine hydrochloride, were thoroughly studied. The constants of binding of Mg2+ to mutant enzymes in the absence of the substrate and to enzyme-substrate complexes were determined. The introduction of amino acid substitutions was shown to stabilize the protein globule. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

Metal-free PPi activates hydrolysis of MgPPi by an Escherichia coli inorganic pyrophosphatase.

Soluble inorganic pyrophosphatase from Escherichia coli (E-PPase) is a hexamer forming under acidic conditions the active trimers. We have earlier found that the hydrolysis of a substrate (MgPP(i)) by the trimers as well as a mutant E-PPase Asp26Ala did not obey the Michaelis-Menten equation. To explain this fact, a model has been proposed implying the existence of, aside from an active site, an effector site that can bind PP(i) and thus accelerate MgPP(i) hydrolysis. In this paper, we demonstrate that the noncompetitive activation of MgPP(i) hydrolysis by metal-free PP(i) can also explain kinetic features of hexameric forms of both the native enzyme and the specially obtained mutant E-PPase with a substituted residue Glu145 in a flexible loop 144-149. Aside from PP(i), its non-hydrolyzable analog methylene diphosphonate can also occupy the effector site resulting in the acceleration of the substrate hydrolysis. Our finding that two moles of [32P]PP(i) can bind with each enzyme subunit is direct evidence for the existence of the effector site in the native E-PPase.

Active dimeric form of inorganic pyrophosphatase from Escherichia coli.

A dimeric form can be obtained from native hexameric Escherichia coli inorganic pyrophosphatase (E-PPase) by destroying the hydrophobic intersubunit contacts, and it has been shown earlier to consist of the subunits of different trimers. The present paper is devoted to the kinetic characterization of such a "double-decked" dimer obtained by the dissociation of either the native enzyme or the mutant variant Glu145Gln. The dimeric form of the native inorganic pyrophosphatase was shown to retain high catalytic efficiency that is in sharp contrast to the dimers obtained as a result of the mutations at the intertrimeric interface. The dimeric enzymes described in the present paper, however, have lost the regulatory properties, in contrast to the hexameric and trimeric forms of the enzyme.

[Hexameric, trimeric, dimeric, and monomeric forms of inorganic pyrophosphatase from Escherichia coli]. / Geksamernaia, trimernaia, dimernaia i monomernaia formy neorganicheskoi pirofosfatazy Escherichia coli.

The conditions were found for obtaining trimeric, dimeric, and monomeric forms of the Escherichia coli inorganic pyrophosphatase from its native hexameric form. Interconversions of the oligomers were studied, and rate constants for their dissociation and association were determined. All forms were found to be catalytically active, with the activity decreasing in the order: hexamer-trimer-dimer-monomer. The activity of trimeric and dimeric forms was high enough to study and to compare their catalytic properties. The monomeric form of the enzyme was unstable.

The role of Asp42 in Escherichia coli inorganic pyrophosphatase functioning.

Excess of Mg2+ ions is known to inhibit the soluble inorganic pyrophosphatases (PPases). In contrast, the mutant Escherichia coli inorganic pyrophosphatase Asp42-->Asn is three times more active than native and retains its activity at high Mg2+ concentration. In this paper, another two mutant variants with Asp42 replaced by Ala or Glu were investigated to characterize the role of Asp42 in catalysis. pH-independent kinetic parameters of MgPPi hydrolysis and the dissociation constants for the activating and inhibitory Mg2+ ions were calculated. It was shown that Mg2+ inhibition of MgPPi hydrolysis by native PPase exhibited uncompetitive kinetics under the saturating substrate concentration. All three substitutions of Asp42 lead to a sharp decrease of inhibitory Mg2+ affinity to the enzyme. These findings allow determination of the sites of inhibitory and substrate Mg2+ ions binding to PPase. Common features of these mutants allow the conclusion that the function of Asp42 is to accurately coordinate the residues implicated in the substrate and the inhibitory Mg2+ ion binding to PPase active site. Structural analysis of PPase complexed with Mg2+ compared with PPase complexed with Mn2+ and reaction products confirms this supposition.

[Inhibition of inorganic pyrophosphatase from Escherichia coli with inorganic phosphate]. / Ingibirovanie neorganicheskoi pirofosfatazy Escherichia coli neorganicheskim fosfatom.

The interaction of inorganic pyrophosphatase from E. coli with inorganic phosphate (Pi) was studied in a wide concentration range of phosphate. The apoenzyme gives two inactive compounds with Pi, a product of phosphorylation of the carboxylic group of the active site and a stable complex, which can be detected in the presence of the substrate. The phosphorylation occurs when Pi is added on a millimole concentration scale, and micromole concentrations are sufficient for the formation of the complex. The formation of the phosphorylated enzyme was confirmed by its sensitivity to hydroxylamine and a change in the properties of the inactive enzyme upon its incubation in alkaline medium. The phosphorylation of pyrophosphatase and the formation of the inactive complex occur upon interaction of inorganic phosphate with different subsites of the enzyme active sites, which are connected by cooperative interactions.

The structures of Escherichia coli inorganic pyrophosphatase complexed with Ca(2+) or CaPP(i) at atomic resolution and their mechanistic implications.

Two structures of Escherichia coli soluble inorganic pyrophosphatase (EPPase) complexed with calcium pyrophosphate (CaPP(i)-EPPase) and with Ca(2+) (Ca(2+)-EPPase) have been solved at 1.2 and 1.1 A resolution, respectively. In the presence of Mg(2+), this enzyme cleaves pyrophosphate (PP(i)) into two molecules of orthophosphate (P(i)). This work has enabled us to locate PP(i) in the active site of the inorganic pyrophosphatases family in the presence of Ca(2+), which is an inhibitor of EPPase.Upon PP(i) binding, two Ca(2+) at M1 and M2 subsites move closer together and one of the liganded water molecules becomes bridging. The mutual location of PP(i) and the bridging water molecule in the presence of inhibitor cation is catalytically incompetent. To make a favourable PP(i) attack by this water molecule, modelling of a possible hydrolysable conformation of PP(i) in the CaPP(i)-EPPase active site has been performed. The reasons for Ca(2+) being the strong PPase inhibitor and the role in catalysis of each of four metal ions are the mechanistic aspects discussed on the basis of the structures described.

Active site interactions in oligomeric structures of inorganic pyrophosphatases.

Recent progress in studies of the mode of action of cytoplasmic inorganic pyrophosphatases is mainly due to the analysis of a dozen and a half structures of the apoenzyme, its complexes, and mutants. However, despite considerable research on the mechanism of action of these enzymes, many important problems remain unclear. Among them is the problem of active site interactions in oligomeric structures and their role in catalysis; this review focuses on this problem. The abundant experimental data requires generalization and comprehensive analysis. A characteristic feature of the spatial structure of inorganic pyrophosphatases is a flexible system of noncovalent interactions between protein groups penetrating the whole molecule of the oligomeric enzyme. Binding of metal ions, sulfate (an analog of the product of the enzymatic reaction), and affinity phosphorus-containing inhibitors at the active site or site-directed mutagenesis induce rearrangements in the set of hydrogen and ionic interactions, which change active site properties and in some instances, cause molecule asymmetry. In the trimeric form of Escherichia coli pyrophosphatase obtained by dissociation of a hexamer, active sites also interact with each other, which is manifested by negative cooperativity upon substrate binding. The association of trimers into the hexamer leads to perfect organization of active sites and to their coordinated functioning, probably due to the restoration of communication channels between the trimers.

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.

Stabilization of the enzyme--substrate complex of the mutant Asp-67Asn inorganic pyrophosphatase from Escherichia coli by fluoride ions.

Magnesium-supported PPi hydrolysis by the mutant Asp-67Asn E. coli pyrophosphatase at saturating PPi and metal-activator concentrations in the presence of NaF is followed by a gradual decrease in the initial rate of PPi hydrolysis. The reaction occurs in two steps: first a complex containing enzyme, pyrophosphate, magnesium, and fluoride ions is immediately formed, then its conformation changes slowly. This enzyme--substrate complex stabilized by fluoride is partially active and can be isolated by the removal of excess fluoride by gel-filtration.

Three-dimensional structures of mutant forms of E. coli inorganic pyrophosphatase with Asp-->Asn single substitution in positions 42, 65, 70, and 97.

The three-dimensional structures of four mutant E. coli inorganic pyrophosphatases (PPases) with single Asp-->Asn substitutions at positions 42, 65, 70, and 97 were solved at 1.95, 2.15, 2.10, and 2.20 A resolution, respectively. Asp-42-->Asn and Asp-65-->Asn mutant PPases were prepared as complexes with sulfate--a structural analog of phosphate, the product of enzymatic reaction. A comparison of mutant enzymes with native PPases revealed that a single amino acid substitution changes the position of the mutated residue as well as the positions of several functional groups and some parts of a polypeptide chain. These changes are responsible for the fact that mutant PPases differ from the native ones in their catalytic properties. The sulfate binding to the mutant PPase active site causes molecular asymmetry, as shown for the native PPase earlier. The subunit asymmetry is manifested in different positions of sulfate and several functional groups, as well as changes in packing of hexamers in crystals and in cell parameters.

Changes in E. coli inorganic pyrophosphatase structure induced by binding of metal activators.

The three-dimensional structures of E. coli inorganic pyrophosphatase (PPase) and its complexes with Mn2+ in a high affinity site and with Mg2+ in high and low affinity sites determined by authors in 1994-1996 at 1.9-2.2 A resolution are compared. Metal ion binding initiates the shifts of alpha-carbon atoms and of functional groups and rearrangement of non-covalent interaction system of hexameric enzyme molecule. As a result, the apoPPase with six equal subunits turns after Mg2+ binding into the structure with three types of subunits distinguished by structure and occupance of the low affinity Mg2+ site. Induced asymmetry reflects the subunit interactions and cooperativity between Mg2+ binding sites. These molecular rearrangements are structural basis to account for special features of the enzyme behavior and to propose one of the pathways for enzymatic activity regulation of constitutive PPases in vivo.

Crystal structure of holo inorganic pyrophosphatase from Escherichia coli at 1.9 A resolution. Mechanism of hydrolysis.

Crystalline holo inorganic pyrophosphatase from Escherichia coli was grown in the presence of 250 mM MgCl2. The crystal structure has been solved by Patterson search techniques and refined to an R-factor of 17.6% at 1.9 A resolution. The upper estimate of the root-mean-square error in atomic positions is 0.26 A. These crystals belong to space group P3(2)21 with unit cell dimensions a = b = 110.27 A and c = 78.17 A. The asymmetric unit contains a trimer of subunits, i.e., half of the hexameric molecule. In the central cavity of the enzyme molecule, three Mg2+ ions, each shared by two subunits of the hexamer, are found. In the active sites of two crystallographically independent subunits, two Mg2+ ions are bound. The second active site Mg2+ ion is missing in the third subunit. A mechanism of catalysis is proposed whereby a water molecule activated by a Mg2+ ion and Tyr 55 play essential roles.

[Irreversible specific inhibition of E. coli inorganic pyrophosphatase with amines]. / Neobratimoe spetsificheskoe ingibirovanie neorganicheskoi pirofosfatazy E. coli aminami.

An unusually high reactivity of the carboxyl groups of the active site of E. coli inorganic pyrophosphatase towards amines was shown. Amino acid esters and other amines are specific irreversible inhibitors of the enzyme. The reaction involves the formation of an enzyme-inhibitor complex followed by the chemical modification of dicarboxylic amino acid residues. It is assumed that the binding of the positively charged inhibitor occurs at the binding site of cations-activators.

Effect of D42N substitution in Escherichia coli inorganic pyrophosphatase on catalytic activity and Mg2+ binding.

Asp-42 located in the active site of E. coli inorganic pyrophosphatase (PPase) has been substituted by Asn by site-directed mutagenesis. This resulted in a 3-fold increase in hydrolytic activity measured under optimal conditions, a 15.5-fold increase in the Km value and retention of the pK values of groups for enzyme and enzyme-substrate complex. The active site of the enzyme contains 4 metal binding centers (I-IV) [Harutyunyan et al. (1996) Eur. J. Biochem., in press]. Asp-42 is located near centers II and IV. The D42N replacement had no effect on Mg2+ binding with center II. At the same time, occupation of center IV eliminates the inhibition of inorganic pyrophosphate hydrolysis by high Mg2+ concentrations typical of wild-type PPase. It is proposed that the increase in activity and decrease in affinity for substrate of the D42N PPase results from changes in Mg2+ binding to center IV. The Mg2+ binding centers of E. coli PPase are lined up in filling order.

Mg2+ activation of Escherichia coli inorganic pyrophosphatase.

Further refinement of X-ray data on Escherichia coli inorganic pyrophosphatase [Oganessyan et al. (1994) FEBS Lett. 348, 301-304] to 2.2 A reveals a system of noncovalent interactions involving Tyr55 and Tyr141 in the active site. The pKa for one of the eight Tyr residues in wild-type pyrophosphatase is as low as 9.1 and further decreases to 8.1 upon Mg2+ binding, generating characteristic changes in the absorption spectrum. These effects are lost in a Y55F but not in a Y141F variant. It is suggested that the lower-affinity site for Mg2+ in the enzyme is formed by Tyr55 and Asp70, which are in close proximity in the apo-enzyme structure.