Tatjana I. Nazarova

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inorganic pyrophosphatase of Escherichia coli, identified as evolutionarily conserved residues of the active site, have been replaced by asparagine.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 κ app value by approx. 2–3 orders of magnitude. At the same time, the K m values changed only slightly. Only minor changes take place in the pK values of the residues essential for both substrate binding and catalysis. All mutant variants have practically the same affinity to Mg2+ as the wild‐type pyrophosphatase.

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.

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 Å resolution using Pattersons search technique and refined to an R‐factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (α3)2 into α3′α3″. The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re‐arrangements for pyrophosphatase function is discussed.

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 K m 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 Å 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.

The phosphorylation of yeast inorganic pyrophosphatase and formation of stoichiometric amounts of enzyme-bound pyrophosphate

1. Introduction Baker’s yeast inorganic pyrophosphatase (EC 3.6.1 .I) is able to bind orthophosphate with a dissoci- ation constant of the order of 1 mM [l-3]. Despite the moderate strength of the binding, it results in formation of a chemical bond of an acyl phosphate type [4]. Since phosphate is the product of PPt hydrolysis and the substrate in the reverse reaction, one could have supposed the phosphorylated enzyme to be an obligatory catalytic intermediate. However, blocking the active site of the enzyme with pyrophos- phate did not eliminate Pi binding [5]. This observa- tion necessitated further studies of the reaction of pyrophosphatase with Pi. They were partly stimulated by the finding of N. N. Vorobjeva in this laboratory that the phosphorylated enzyme can be isolated by gel filtration in the absence of Mg*+. Here, the following features of the enzyme-phos- phate reaction are revealed: (i) A stoichiometric amount of protein-bound PP, forms from Pi in the active site of pyrophos- phatase; (ii) 0.5 mol Pi/m01 of active sites is independently bound in a different centre; (iii) The bound Pi and PPi are rapidly exchanged for medium phosphate in the presence of Mg*+. 2. Materials and methods Inorganic pyrophosphatase with spec. act. 600 IV/ mg at pH 7.2,25”C was isolated from baker’s yeast as

Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO4 2

The three‐dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 Å resolution using Pattersons search technique and refined to an R‐factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (α3)2 into α3′α3″. The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re‐arrangements for pyrophosphatase function is discussed.

The flip-flop mechanism of the phosphorylation of yeast inorganic pyrophosphatase.

1. An active monomeric form of inorganic pyrophosphatase from bakers yeast was prepared by maleylation of the protein at pH 10.5. 2. The dimeric and monomeric pyrophosphatase bound at non-catalytic sites 0.5 and 1.0 mol of slowly dissociating Pi per mol subunit, respectively. This stoichiometry was not affected on active site blockage with PPi. 3. Added Pi accelerated the dissociation of Pi from the dimeric but not monomeric enzyme. 4. Our results indicate a strong interaction to occur between the non-catalytic sites of two subunits of native pyrophosphatase which results in diminished stability of Pi binding to one of them.

Crystal structure ofEscherichia coliinorganic pyrophosphatase complexed with SO42−: Ligand-induced molecular asymmetry

The three‐dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 Å resolution using Pattersons search technique and refined to an R‐factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (α3)2 into α3′α3″. The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re‐arrangements for pyrophosphatase function is discussed.

Site-site interactions and regulation of the activity of inorganic pyrophosphatases

Abstract Inorganic pyrophosphatases of bakers yeast and E. coli are oligomers built of chemically identical subunits. Both enzymes are active in the monomeric state. Each subunit has an active and an allosteric site linked by hetero- and homotropic interactions. These interactions are responsible for the regulation of pyrophosphatase activity. Typical of these enzymes is the autocatalytic phosphorylation of the allosteric or active site and the regeneration of the initial enzyme via dephosphorylation in the process of the conformational change of the enzyme.