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.

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.

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.

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.

[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.

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.

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.

Effectory site in Escherichia coli inorganic pyrophosphatase is revealed upon mutation at the intertrimeric interface.

Escherichia coli inorganic pyrophosphatase (E-PPase) is a homohexamer formed from two trimers related by a two-fold axis. The residue Asp26 participates in intertrimeric contacts. Kinetics of MgPPi hydrolysis by a mutant Asp26Ala E-PPase is found to not obey Michaelis-Menten equation but can be described within the scheme of activation of hydrolysis by a free PPi binding at an effectory subsite.

[Hexameric, trimeric, dimeric, and monomeric forms of inorganic pyrophosphatase from 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 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.

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.

[Inhibition of inorganic pyrophosphatase from Escherichia coli with inorganic phosphate].

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.

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.

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.

Interaction of Escherichia coli inorganic pyrophosphatase active sites.

Escherichia coli inorganic pyrophosphatase (PPase) is a hexamer of identical subunits. This work shows that trimeric form of PPase exhibits the interaction of the active sites in catalysis. Some trimer subunits demonstrate high substrate binding affinity typical for hexamer whereas the rest of subunits reveal more than 300-fold substrate affinity decrease.

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.

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.

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.

Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO4(2-). Ligand-induced molecular asymmetry.

The three-dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 A resolution using Patterson's search technique and refmed to an R-factor of 19.2%.

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.

[Irreversible specific inhibition of E. coli inorganic pyrophosphatase with amines].

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.

Escherichia coli inorganic pyrophosphatase: site-directed mutagenesis of the metal binding sites.

Aspartic acids 65, 67, 70, 97 and 102 in the inorganic pyrophosphatase of Escherichia coli, identified as evolutionarily conserved residues of the active site, have been replaced by asparagine. Each mutation was found to decrease the k(app) value by approx. 2-3 orders of magnitude.

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.

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.

X-ray crystallographic studies of recombinant inorganic pyrophosphatase from Escherichia coli.

An E. coli inorganic pyrophosphatase overproducer and a method for a large-scale production of the homogeneous enzyme are described. The inorganic pyrophosphatase was crystallized in the form containing one subunit of a homohexameric molecule per asymmetric unit: space group R32, a = 110.