Structural and kinetic features of family I inorganic pyrophosphatase from Vibrio cholerae.

In this paper, kinetic properties of a soluble inorganic pyrophosphatase of family I from Vibrio cholerae (V-PPase), intestinal pathogen and causative agent of human cholera, are characterized in detail, and the crystal structure of a metal-free enzyme is reported. Hydrolytic activity of V-PPase has been studied as a function of pH, concentration of metal cofactors (Mg2+ or Mn2+), and ionic strength. It has been found that, despite the high conservation of amino acid sequences for the known bacterial PPases of family I, V-PPase differs from the other enzymes of the same family in a number of parameters. Dissociation constants of V-PPase complexed with Mg2+ or Mn2+ were essentially the same as for Escherichia coli PPase (E-PPase). However, the pH optimum of MgPP(i) hydrolysis by V-PPase was shifted to more alkaline pH due to higher values of the pK(a) of ionizable groups for both the free enzyme and the enzyme-substrate complex. The stability of a hexameric form of V-PPase has been studied as a function of pH. The corresponding pK(a) of a group that controls the stability of the hexamer at pH below 6 (pK(a) = 4.4) was significantly lower than in the other hexameric PPases. The crystal structure reported here is analyzed and compared with the structure of E-PPase. The location of amino acid residues that differ in V-PPase and E-PPase is discussed. Since V-PPase has been found to retain its hydrolytic activity in high ionic strength media, the observed structural and kinetic features are analyzed in view of the possible osmoadaptation of this protein.

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.

Fluoride inhibition of inorganic pyrophosphatase. II. Isolation and characterization of a covalent intermediate between enzyme and entire substrate molecule.

A presumed pyrophosphoryl-enzyme intermediate of the reaction catalyzed by bakers' yeast inorganic pyrophosphatase pyrophosphate phosphohydrolase, EC 3.6.1.1) has been isolated using fluoride-mediated inactivation of the enzyme during catalysis. The analysis of the F--inactivated pyrophosphatase revealed the presence of one molecule of PPi and one atom of fluoride per active site. The incubation of the inactivated enzyme at 25 degrees C and pH 7.2 resulted in gradual recovery of catalytic activity and concomitant removal of PPi by a first-order reaction with tau1/2 of 1 h. The digestion of the F--treated pyrophosphatase with pepsin yielded phosphorous-containing peptides, which were reduced with NaBH4 and gave homoserine and homoserine lactone after acid hydrolysis. This suggests that the PPi residue is linked to the protein through a bond of an acyl phosphate type involving the beta-COOH function of aspartic acid. Together with the results of the kinetic studies of fluoride inhibition of pyrophosphatase reported in accompanying papers, these findings strongly indicate that the enzyme-substrate compound stabilized by fluoride is a transient of the catalytic reaction.

The essential activated carboxyl group of inorganic pyrophosphatase.

1. A carboxyl group of high reactivity has been found in inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from yeast. This group interacts with agents which react neither with carboxyl groups of low molecular weight compounds nor with other carboxyl groups of the protein. 2. The reaction of this activated carboxyl group with inorganic phosphate, hydroxylamine, N-methyl- and O-methylhydroxylamines, and glycine methyl ester has been studied. 3. Homoserine and homoserine lactone were found in the hydrolyzate of phosphorylated and NaBH4-reduced pyrophosphatase, indicating that an aspartyl residue is phosphorylated. 4. Hydroxylamine and other nucleophilic agents cause inactivation of pyrophosphatase as a result of interaction with a carboxyl group. Both diaminobutyric and diaminopropionic acids were seen in the acid hydrolyzate of the protein treated with hydroxylamine and subjected to rearrangement in the presence of carbodiimide. 5. The ways in which the activation of a carboxyl group in the enzyme is achieved and the presumed mechanism of action of inorganic pyrophosphatase are discussed.

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.

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.

Expression of Saccharomyces cerevisiae inorganic pyrophosphatase in Escherichia coli.

A segment of DNA encoding Saccharomyces cerevisiae inorganic pyrophosphatase (ppa gene) was amplified by the polymerase chain reaction. The pSCH1 and pSCB6 plasmids containing the ppa gene were obtained. Transformation of the E. coli BL21 strain with the resulting recombinant plasmids and selection of clones having extremely high expression of inorganic pyrophosphatase (PPase) were carried out. Superproduction of recombinant S. cerevisiae PPase up to 50% of the total bacterial protein was achieved. The enzyme was readily obtained and purified to homogeneity with the use of a simple purification technique. This work is the first description of S. cerevisiae PPase superproducer creation.

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 = 110.4 A, c = 76.8 A. The electron density map to 2.5 A resolution phased with Eu- and Hg-derivatives (figure of merit, = 0.51) was improved by the solvent flattening procedure ( = 0.77). The course of the polypeptide chain and the secondary structure elements, intersubunit contacts and positions of the active sites were characterized. Homology with S. cerevisiae inorganic pyrophosphatase structure was found.

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.

[Phosphorylation as a method of regulating inorganic pyrophosphatase activity in E. coli. I. Phosphorylation and enzyme activation induced by ATP]. / Fosforilirovanie kak sposob reguliatsii aktivnosti neorganicheskoi pirofosfatazy E. coli. I. Fosforilirovanie i aktivatsiia fermenta pod deistviem ATP.

ATP phosphorylates the regulatory center of E. coli inorganic pyrophosphatase with the resultant 1,5-fold increase in the activity of the enzyme. The maximal incorporation of the ATP gamma-group into pyrophosphatase is 3 moles per mole of the protein. Pi likewise phosphorylates the enzyme regulatory center and lowers the pyrophosphatase activity by 10-15%. The ATP- and Pi-mediated phosphorylation processes are interrelated; ATP prevents phosphorylation by Pi and brings about rapid dephosphorylation of Pi-modified protein.

[Phosphorylation as a method of regulating inorganic pyrophosphatase activity in E. coli. II. Identification of the types of chemical bonds between phosphate and the enzyme]. / Fosforilirovanie kak sposob reguliatsii aktivnosti neorganicheskoi pirofosfatazy E. coli. II. Identifikatsiia tipa khimicheskikh sviazei fosfata s fermentom.

A bond formed by phosphate with Pi-phosphorylated pyrophosphatase from E. coli was found to be labile in acidic and alkaline media and to be rapidly cleaved by hydroxylamine at neutral pH. N-Methylhydroxylamine modifies also activated carboxyl groups of the enzyme. Interaction of inorganic pyrophosphatase with ATP produces an alkali-resistant phosphoamide bond. A phosphorylated amino acid, identified as phosphohistidine, was isolated from the alkaline hydrolyzate of the ATP-phosphorylated pyrophosphatase.

Metal cofactors play a dual role in Mycobacterium tuberculosis inorganic pyrophosphatase.

Inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) is one of the possible targets for the rational design of anti-tuberculosis agents. In this paper, functional properties of this enzyme are characterized in the presence of the most effective activators--Mg2+ and Mn2+. Dissociation constants of Mt-PPase complexed with Mg2+ or Mn2+ are essentially similar to those of Escherichia coli PPase. Stability of a hexameric form of Mt-PPase has been characterized as a function of pH both for the metal-free enzyme and for Mg2+- or Mn2+-enzyme. Hexameric metal-free Mt-PPase has been shown to dissociate, forming monomers at pH below 4 or trimers at pH from 8 to 10. Mg2+ or Mn2+ shift the hexamer-trimer equilibrium found for the apo-Mt-PPase at pH 8-10 toward the hexameric form by stabilizing intertrimeric contacts. The pK(a) values have been determined for groups that control the observed hexamer-monomer (pK(a) 5.4), hexamer-trimer (pK(a) 7.5), and trimer-monomer (pK(a) 9.8) transitions. Our results demonstrate that due to the non-conservative amino acid residues His21 and His86 in the active site of Mt-PPase, substrate specificity of this enzyme, in contrast to other typical PPases, does not depend on the nature of the metal cofactor.

ATP as effector of inorganic pyrophosphatase of Escherichia coli. The role of residue Lys112 in binding effectors.

It has been shown that PP(i), methylenediphosphonate, and ATP act as effectors of Escherichia coli inorganic pyrophosphatase (E-PPase), and that they compete for binding at the allosteric regulatory site. On the basis of chemical modification and computer modeling of a structure of the enzyme-ATP complex, a number of amino acid residues presumably involved in binding effectors has been revealed. Mutant variants Lys112Gln, Lys112Gln/Lys148Gln, and Lys112Gln/Lys115Ala of E-PPase have been obtained, as well as a modified variant of wild type E-PPase ((Ad)wt PPase) with a derivative of ATP chemically attached to the amino group of Lys146. Kinetic properties of these variants have been investigated and compared to the earlier described variants Lys115Ala, Arg43Gln, and Lys148Gln. Analysis of the data confirms the proposed location of an effector binding site in a cluster of positively charged amino acid residues including the side chains of Arg43, Lys146 (subunit A), Lys112, and Lys115 (subunit B). Lys112 is supposed to play a key role in forming contacts with the phosphate groups of the three studied effectors.

ATP as effector of inorganic pyrophosphatase of Escherichia coli. Identification of the binding site for ATP.

The interaction of Escherichia coli inorganic pyrophosphatase (E-PPase) with effector ATP has been studied. The E-PPase has been chemically modified with the dialdehyde derivative of ATP. It has been established that in the experiment only one molecule of effector ATP is bound to each subunit of the hexameric enzyme. Tryptic digestion of the adenylated protein followed by isolation of a modified peptide by HPLC and its mass-spectrometric identification has showed that it is an amino group of Lys146 that undergoes modification. Molecular docking of ATP to E-PPase indicates that the binding site for effector ATP is located in a cluster of positively charged amino acid residues proposed earlier on the basis of site-directed mutagenesis to participate in binding of effector pyrophosphate. Molecular docking also reveals several other amino acid residues probably involved in the interaction with effectors.

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.

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.

[Some features of methylpyrophosphate hydrolysis by yeast inorganic pyrophosphatase]. / Osobennosti gidroliza metilphirofosfata neorganicheskoi pirofosfatazoi iz drozhzhei.

The interaction of yeast inorganic pyrophosphatase with methylpyrophosphate was studied. In the presence of Mg2+ the rate of hydrolysis of the methylpyrophosphate-Zn2+ complex by the enzyme was shown to decrease. This was accompanied by competition of Zn2+ and Mg2+ for one site of Me2+ binding on the enzyme. The kinetics of combined hydrolysis of zinc methylpyrophosphate and zinc pyrophosphate were studied. It was found that both substrates are hydrolyzed at the same active site of the enzyme. Free methylpyrophosphate when bound to a specific phosphorylation site on the enzyme surface accelerated magnesium pyrophosphate hydrolysis. Some kinetic parameters of this hydrolysis were determined.