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.     

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 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.     

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.     

The effect of oxidized glutathione and its pharmacological analogue, glutoxim, on intracellular Ca2+ concentration in macrophages

The effect of oxidized glutathione (GSSG) and its pharmacological analogue, glutoxim, on intracellular Ca2+ concentration in rat peritoneal macrophages was investigated using Fura-2AM microfluorimetry. It was shown that both GSSG and glutoxim increased intracellular Ca2+ concentration inducing Ca(2+)-mobilization from thapsigargin-sensitive Ca(2+)-stores and subsequent Ca2+ entry into macrophages from external medium. Dithiothreitol, which reduces S-S-bonds in proteins, completely prevented or reversed the increase in the intracellular Ca2+ concentration induced by GSSG or glutoxim. It suggests that the increase in the intracellular Ca2+ concentration induced by GSSG or glutoxim can be mediated by their interactions with functionally important SH-groups of proteins involved in Ca(2+)-signaling. Two structurally different tyrosine kinase inhibitors, genistein and methyl-2,5-dihydroxycinnamate, prevented or completely reversed the increase in the intracellular Ca(2+)-concentration induced by GSSG or glutoxim. On the contrary, tyrosine phosphatase inhibitor, Na orthovanadate, enhanced the increase in the intracellular Ca2+ concentration evoked by oxidizing agents. The data suggest that tyrosine kinases and tyrosine phosphatases are involved in regulatory effects of GSSG and glutoxim on the intracellular Ca2+ concentration in macrophages.     

Genome-wide functional annotation of dual-specificity protein- and lipid-binding modules that regulate protein interactions

Emerging evidence indicates that membrane lipids regulate protein networking by directly interacting with protein-interaction domains (PIDs). As a pilot study to identify and functionally annodate lipid-binding PIDs on a genomic scale, we performed experimental and computational studies of PDZ domains. Characterization of 70 PDZ domains showed that ~40% had submicromolar membrane affinity. Using a computational model built from these data, we predicted the membrane-binding properties of 2,000 PDZ domains from 20 species. The accuracy of the prediction was experimentally validated for 26 PDZ domains. We also subdivided lipid-binding PDZ domains into three classes based on the interplay between membrane- and protein-binding sites. For different classes of PDZ domains, lipid binding regulates their protein interactions by different mechanisms. Functional studies of a PDZ domain protein, rhophilin 2, suggest that all classes of lipid-binding PDZ domains serve as genuine dual-specificity modules regulating protein interactions at the membrane under physiological conditions.     

The involvement of actin cytoskeleton in glutoxim and molixan effect on intracellular Ca<Superscript>2+</Superscript>-concentration in macrophages

Glutoxim and molixan belong to new generation of disulfide-containing drugs with immunomodulatory, hepatoprotective and hemopoetic effect on cells. Using Fura-2AM microfluorimetry, two structurally distinct actin filament disrupters latrunculin B and cytochalasin D, and calyculin A, which causes actin filaments condensation under plasmalemma, we have shown the involvement of actin cytoskeleton in the intracellular Ca²⁺-concentration increase induced by glutoxim or molixan in rat peritoneal macrophages. Morphological data obtained with the use of rhodamine-phalloidine demonstrated that glutoxim and molixan cause the actin filaments reorganization in rat peritoneal macrophages.     

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 arachidonic and other fatty acids on the intracellular calcium concentration and calcium signaling in peritoneal macrophages

Effects of arachidonic and other fatty acids on the intracellular Ca2+ concentration ([Ca2+]i) in rat peritoneal macrophages was investigated. It has been shown that cis-polyunsaturated arachidonic and linoleic induce a significant and dose-dependent increase in [Ca2+]i, which is due to depletion of thapsigargin-sensitive Ca2+ store and to stimulation of Ca2+ entry from the extracellular medium. Pharmacological characteristics of Ca2+ entry induced by arachidonic acid appeared to be similar to those of store-dependent Ca2+ entry activated by thapsigargin or cyclopiazonic acid; Ca2+ entry is attenuated by the same Ca2+ channel inhibitors, by tyrosine kinase inhibitor genistein and epoxygenase inhibitor proadifen. Cis-monounsaturated oleic and saturated myristic acids appeared to be less effective and induced only a slight increase in [Ca2+]i at much higher concentrations. Arachidonic and other fatty acids can also stimulate Ca(2+)-ATPase in the macrophage plasma membrane. The data are compatible with the important role played by arachidonic and other free fatty acids in the regulation of [Ca2+]i in peritoneal macrophages.