Vera Sklyankina

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.

The quaternary structure of E.coli inorganic pyrophosphatase is not required for catalytic activity

Inorganic pyrophosphatase Subunit Quaternary structure Kinetic analysis

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. This fact indicates the appearance of negative cooperativity of trimer subunits upon substrate binding. Association of the wild‐type (WT) trimer with catalytically inactive, but still substrate binding mutant trimer into hexameric chimera restores the high activity of the first trimer, characteristic of trimer incorporated in the hexamer of WT PPase. Interaction of PPase active sites suggests that there are pathways for information transmission between the active sites, providing the perfect organization and concerted functioning of the hexameric active sites in catalysis.

Half‐of‐the‐sites reactivity of inorganic pyrophosphatase from yeast is the result of induced asymmetry

Baker’s yeast pyrophosphatase (EC 3.6.1.1) displays half-of-the-sites reactivity with respect to the inhibitors, phosphoric acid monoesters [ 1,2]. The enzyme catalyzes the hydrolysis and synthesis of inorganic pyrophosphate. It is a dimeric protein which consists of identical subunits, each containing an active centre. This enzyme is one of a growing list of proteins which display half-of-the-sites behaviour. The half-of-the-sites reactivity in proteins can be explained on the basis of a number of mechanisms [3]. Taking into account that the two subunits of the pyrophosphatase molecule are identical in primary structure and that the catalytic sites are distant from each other [4], the half-of-the-sites behaviour in pyrophosphatase is best explained in terms of the so-called pre-existing asymmetry or induced asymmetry models. In the former model, the oligomer consists of two conformationally different classes of subunits, which differ in their reactivity towards a modifying agent. According to the simple version of this model, whatever happens at one subunit has no influence on events that might occur on the neighbouring subunit. In the model of induced asymmetry, the subunits are conformationally identical in the native enzyme but modification of any subunit induces a conformational change in its neighbour with a resultant Half-of-the-sites reactivity

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.

Tyrosine-89 is important for enzymatic activity of S. cerevisiae inorganic pyrophosphatase

7‐Chloro‐4‐nitro‐benzofurazan selectively modifies one PPase Tyr residue per subunit and lowers the enzyme activity. Hydrolysis of the modified protein by trypsin and then by chymotrypsin produces the 82–89 peptide which possesses modified Tyr‐89. Substrate analog (CaPPi) and the product of the enzyme reaction, MgP1, protect the enzyme against inactivation. Ions of metal‐activators (Mg2+, Zn2+) exert no influence on the inactivation rate. On the contrary, the Ca2+‐inhibitor of the enzyme accelerates the reaction by binding to the high‐affinity site, and effectively decreases it when Ca2+ binds to both sites. Mg2+ competes with Ca2+ for one binding site, which is the low affinity site for Mg2+ and the high‐affinity site for Ca2+. The Ca2+ saturation of the high‐affinity site decreases the pK 2 of Tyr‐89, probably due to direct coordination between Tyr and Ca2+. The observed properties of Tyr‐89 modification enable us to propose that Tyr‐89 serves as a proton donor for phosphate releasing during enzymatic hydrolysis of pyrophosphate. The Ca2+ inhibitory effect on the enzyme activity may be due to the existence of a Tyr‐89 bond in the Cn2+ pyrophosphatase complex.

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. 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.

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.