Effect of divalent metal ions on soluble and membrane-bound alkaline phosphatase activity in suckling rat intestine.

EDTA treatment of intestinal brush border membranes (BBM) and epithelial cell supernatant completely inhibited alkaline phosphatase (AP) activity in suckling rat intestine. AP activity was fully regained upon dialysis of the preparations against Zn2+ and to a lesser extent against Co2+, Ca2+ and Mn2+ ions. Other metal ions (Cd2+ and Mg2+) tested were essentially ineffective in restoring the enzyme activity. Considerable differences were observed in kinetic characteristics of the membrane-bound and soluble AP activities in response to various metal ions. There were apparent differences in Km, Vmax, energy of activation (Ea) and thermal stability of the soluble and membrane-bound AP activities, after metal ion substitutions. Nearly 35% AP activity was solubilized on sodium dodecyl sulphate treatment of brush borders (membrane protein: detergent ratio 1:3; w/w). Dialysis of detergent solubilized BBM against different metal ions reconstituted AP activity in the particulate fraction: the order of effectiveness was Zn greater than Ca greater than Mn greater than Co. The kinetic properties of the reconstituted AP were essentially similar to the non-integrated enzyme activity in response to various divalent metal ions examined. But there were apparent differences in Km, Vmax, Ea and thermal stability of the reconstituted AP activity compared to native brush border enzyme. The results suggest the unique requirement of Zn ions for stability and catalytic activity of the soluble and membrane-bound AP activity in suckling rat intestine.     

Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity

The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a k(cat) of 16 s(-1) and a K(m) of 175 microM at a pH optimum of 8.0 when assayed at 25 degrees C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum k(cat) of 100 s(-1), with a K(m) of 93 microM at 65 degrees C. Activity was stable at 65 degrees C for >24 h and at 90 degrees C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer.     

Characterization of a Highly Thermostable Alkaline Phosphatase from the Euryarchaeon Pyrococcus abyssi

This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70°C, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105°C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiol-reducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intra- or interchain disulfide bonds. In addition, P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.     

Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme

The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca²⁺, Mg²⁺, Mn²⁺, and Sr²⁺ have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed.     

Divalent metal ion effects on a mutant histidinol phosphate phosphatase from Salmonella typhimurium

The effect of divalent metal ions on the activity of a mutant histidinol phosphate phosphatase has been studied. The enzyme was isolated from strain TA387, a mutant of Salmonella typhimurium with a nonsense lesion near the midpoint of the bifunctional hisB gene. Mn²⁺, Mg²⁺, Co²⁺, and Zn²⁺ shift the optimal pH of phosphatase activity to 6.5 while Be²⁺ and Ca²⁺ have no effect on the shape of the pH profile. In the absence of divalent metal ions, the pH optimum is 7.5. Four Me²⁺ ions, Mn²⁺, Co²⁺, Zn²⁺, and Fe²⁺ decreased the K_m of histidinol phosphate at pH 6.5 from 5.5 mm (without Me²⁺) to 0.14 mm. Ni²⁺ and Be²⁺ increased the K_m to 22.2 and 25.0 mm, respectively, and Ca²⁺ and Mg²⁺ had an intermediate effect. Changes in maximal velocity were substantially less, only about 2-fold changes being observed. It was shown that the maximal velocity at optimal pH was the same in the absence and presence of Mn²⁺. Kinetic analysis indicated that there was a rapid equilibrium-ordered addition of Mn²⁺ to the enzyme before the addition of the substrate, histidinol phosphate. A k_imn²⁺ of 4.3 μm was calculated for the metal ion activation at both pH 6.5 and 7.5. Addition of ethyl-enediaminetetracetate (EDTA) strongly inhibited the phosphatase; inhibition could be reversed by addition of several Me²⁺ ions, Mg²⁺ being the most efficient followed by Mn²⁺. Prolonged incubation with EDTA led to irreversible inactivation.     

Identification of brain ecto-apyrase as a phosphoprotein

The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca²⁺ 0.25, Mg²⁺ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 M) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe²⁺, Zn²⁺ or Cu²⁺. Ca²⁺ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg²⁺ or Mn²⁺ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn²⁺ or Ca²⁺ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba²⁺, Zn²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ca²⁺), as well as La³⁺, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca²⁺, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca²⁺.     

Mn2+ and Mg2+ improved sphingomyelinase production by Lactobacillus rhamnosus FTDC 8313 and binding affinity to sphingomyelin for generation of ceramides

The present research aims to optimize the sphingomyelinase (SMase) activity produced by Lactobacillus rhamnosus FTDC 8313 using divalent metal ions via response surface methodology and to further study the effects of the divalent metal ions on SMase activity using molecular modeling approach. This study also aimed to assess the possibility of increasing ceramide levels in vitro on cultured keratinocytes upon treatment with the extracellular extract of the optimized L. rhamnosus FTDC 8313. Using a central composite design, an optimum point of SMase activity (6.54 mU ml^?1) was produced from a combination of 0.65% (w/v) MnSO₄ and 0.82% (w/v) MgSO₄. 3D response surface indicated that the altered availability of the two ions (Mn²⁺ and Mg²⁺) reduced their effects on SMase activity. In addition, the treatment of the HaCaT cells with optimized extracellular extract of L. rhamnosus FTDC8313 significantly increased (P < 0.05) the conversion of sphingomyelin to ceramide as compared to the control. Molecular docking demonstrated that the addition of Mn²⁺ and Mg²⁺ into the active site of SMase improved the binding affinity between the SMase and sphingomyelin based on its free energy of binding as well as the interaction distances between the important catalytic residues Glu53 and His296.     

Nucleotide pyrophosphatase from yeast. The presence of bound zinc.

Nucleotide pyrophosphatase from yeast was inhibited by thiols, o-phenanthroline, 8-hydroxyquinoline, EDTA, and 8-hydroxyquinoline-5-sulfonic acid. The inhibition by chelating agents was time and concentration dependent. Inhibition by EDTA was decreased by complexing the EDTA with metal ions before addition to the enzyme. The effectiveness of the metal ions in preventing inhibition by EDTA paralleled the stability constants of the EDTA-metal complexes. Partial recovery of EDTA-inhibited enzyme activity was achieved with Zn²⁺, Co²⁺, Fe²⁺, and Mn²⁺. Analyses for zinc in the purified enzyme by atomic absorption spectroscopy and by titration with 8-hydroxyquinoline-5-sulfonic acid revealed the presence of approximately 1 g atom/mol of enzyme (M_r 65,000). The data indicate that yeast nucleotide pyrophosphatase is a metalloenzyme in which the zinc plays some role in activity.     

Divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli

The metal-binding properties of the methionyl aminopeptidase from Escherichia coli (MetAP) were investigated. Measurements of catalytic activity as a function of added Co(II) and Fe(II) revealed that maximal enzymatic activity is observed after the addition of only 1 equiv of divalent metal ion. Based on these studies, metal binding constants for the first metal binding event were found to be 0.3 +/- 0.2 microM and 0.2 +/- 0.2 microM for Co(II)- and Fe(II)-substituted MetAP, respectively. Binding of excess metal ions (>50 equiv) resulted in the loss of approximately 50% of the catalytic activity. Electronic absorption spectral titration of a 1 mM sample of MetAP with Co(II) provided a binding constant of 2.5 +/- 0.5 mM for the second metal binding site. Furthermore, the electronic absorption spectra of Co(II)-loaded MetAP indicated that both metal ions reside in a pentacoordinate geometry. Consistent with the absorption data, electron paramagnetic resonance (EPR) spectra of [CoCo(MetAP)] also indicated that the Co(II) geometries are not highly constrained, suggesting that each Co(II) ion in MetAP resides in a pentacoordinate geometry. EPR studies on [CoCo(MetAP)] also revealed that at pH 7.5 there is no significant spin-coupling between the two Co(II) ions, though a small proportion ( approximately 5%) of the sample exhibited detectable spin-spin interactions at pH values > 9.6. EPR studies on [Fe(III)_(MetAP)] and [Fe(III)Fe(III)(MetAP)] also suggested no spin-coupling between the two metal ions. (1)H nuclear magnetic resonance (NMR) spectra of [Co(II)_(MetAP)] in both H(2)O and D(2)O buffer indicated that the first metal binding site contains the only active-site histidine residue, His171. Mechanistic implications of the observed binding properties of divalent metal ions to the MetAP from E. coli are discussed.     

Crystal structure of the single-stranded RNA binding protein HutP from Geobacillus thermodenitrificans

RNA binding proteins control gene expression by the attenuation/antitermination mechanism. HutP is an RNA binding antitermination protein. It regulates the expression of hut operon when it binds with RNA by modulating the secondary structure of single-stranded hut mRNA. HutP necessitates the presence of l-histidine and divalent metal ion to bind with RNA. Herein, we report the crystal structures of ternary complex (HutP–l-histidine–Mg²⁺) and EDTA (0.5 M) treated ternary complex (HutP–l-histidine–Mg²⁺), solved at 1.9 Å and 2.5 Å resolutions, respectively, from Geobacillus thermodenitrificans. The addition of 0.5 M EDTA does not affect the overall metal-ion mediated ternary complex structure and however, the metal ions at the non-specific binding sites are chelated, as evidenced from the results of structural features.     

A limitation of the continuous spectrophotometric assay for the measurement of myo-inositol-1-phosphate synthase activity

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay—the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg²⁺ and is not enhanced by other divalent metal ions (Zn²⁺ and Mn²⁺), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors.     

A chelate complex‐enhanced luminol system for selective determination of Co(II), Fe(II) and Cr(III)

A determination method for Co(II), Fe(II) and Cr(III) ions by luminol‐H₂O₂ system using chelating reagents is presented. A metal ion‐chelating ligand complex with a Co(II) ion and a chelating reagent like ethylenediaminetetraacetic acid (EDTA) produced highly enhanced chemiluminescence (CL) intensity as well as longer lifetime in the luminol‐H₂O₂ system compared to metals that exist as free ions. Whereas free Cu(II) and Pb(II) ions had a strong catalytic effect on the luminol‐H₂O₂ system, significantly, the complexes of Cu(II) and Pb(II) with chelating reagents lost their catalytic activity due to the chelating reagents acting as masking agents. Based on the observed phenomenon, it was possible to determine Co(II), Fe(II) and Cr(III) ions with enhanced sensitivity and selectivity using the chelating reagents of the luminol‐H₂O₂ system. The effects of ligand, H₂O₂ concentration, pH, buffer solution and concentrations of chelating reagents on CL intensity of the luminol‐H₂O₂ system were investigated and optimized for the determination of Co(II), Fe(II) and Cr(III) ions. Under optimized conditions, the calibration curve of metal ions was linear over the range of 2.0 × 10^‐8 to 2.0 × 10^‐5 M for Co(II), 1.0 × 10^‐7 to 2.0 × 10^‐5 M for Fe (II) and 2.0 × 10^‐7 to 1.0 × 10^‐4 M for Cr(III). Limits of detection (3σ/s) were 1.2 × 10^‐8, 4.0 × 10^‐8 and 1.2 × 10^‐7 M for Co(II), Fe(II) and Cr(III), respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Binding of the Activating Ion Co(II) to myo-Inositol Monophosphatase Monitored by Fluorescence and Phosphorescence Spectroscopy

Two extrinsic probes, pyrene-maleimide and eosin-maleimide, were used to label specific SH groups of the enzyme myo-inositol monophosphatase. The fluorescence of pyrene-monophosphatase is enhanced upon addition of the activating metal ions Co(II) and Mg(II). Co(II) ions bind with a dissociation constant of 4 μM, whereas the apparent activation constant K _a is 0.4 mM. Energy transfer measurements demonstrated that the pyrene chromophore, covalently linked to Cys-218, is within 9 Å of the metal ion Tb(III) coordinated to the metal-binding site. The phosphorescence emitted by eosin covalently linked to the protein is quenched by the addition of the activating cations Co(II) and Mg(II). Phosphorescence titrations conducted under anaerobic conditions were used to determine a dissociation constant of approximately 3 μM for the binding of Co(II) ions. The results are consistent with the hypothesis that two activating ions per monomeric subunit participate in the catalytic mechanism. The affinity of the tightly bound ion is at least 100-fold greater than the affinity of the weakly bound ion.     

Effects of metal ions on the hydrolysis of p-nitrophenyl caproate by modified poly(ethylenimine)s

Hydrolysis of nitrophenyl caproate by modified poly(ethylenimine)s containing imidazole moieties is markedly enhanced in the presence of divalent metal ions. The order of effectiveness is Cu(II) > Co(II) > Zn(II) > Ni(II) > Mn(II), with Cu increasing the rate about 20-fold. The acceleration by the metal ion is much greater in the presence of CH₃COO^? or Cl^? as counterions than in the presence of ClO₄^?. Turnover experiments, in which moles of substrate cleaved were in substantial excess of moles of metal present, established the catalytic nature of the effects of the metals. The extent of binding of Cu(II) by the polymers was measured. Maximum accentuation in rate of hydrolysis of nitrophenyl ester was observed for the imidazole-containing polymer when the ratio of imidazole:Cu(II) was 2.75. Some possible mechanisms for the rate enhancement by metal ions are described.     

Kinetic properties of microsomal UDP-glucuronyltransferase. Regulation by metal ions

Treatment of microsomes with EDTA abolishes the stimulation of glucuronidation produced by UDP-N-acetylglucosamine. Addition of divalent metal ions to EDTA-treated microsomes restores the sensitivity of UDP-glucuronyltransferase to UDP-N-acetylglucosamine. Regulation of the activity of this enzyme by UDP-N-acetylglucosamine depends, therefore, on the presence of divalent metal ions. In addition, divalent metals increase the rate of glucuronidation of p-nitrophenol at V_max. The data indicate that metals are essential for the efficient function of UDP-glucuronyltransferase.     

Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis

Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded(ds)-RNA-specific endonuclease with key roles in diverse RNA maturation and decay pathways. E.coli RNase III is a member of a structurally distinct superfamily that includes Dicer, a central enzyme in the mechanism of RNA interference. E.coli RNase III requires a divalent metal ion for activity, with Mg²⁺ as the preferred species. However, neither the function(s) nor the number of metal ions involved in catalysis is known. To gain information on metal ion involvement in catalysis, the rate of cleavage of the model substrate R1.1 RNA was determined as a function of Mg²⁺ concentration. Single-turnover conditions were applied, wherein phosphodiester cleavage was the rate-limiting event. The measured Hill coefficient (n_H) is 2.0 ± 0.1, indicative of the involvement of two Mg²⁺ ions in phosphodiester hydrolysis. It is also shown that 2-hydroxy-4H-isoquinoline-1,3-dione—an inhibitor of ribonucleases that employ two divalent metal ions in their catalytic sites—inhibits E.coli RNase III cleavage of R1.1 RNA. The IC₅₀ for the compound is 14 μM for the Mg²⁺-supported reaction, and 8 μM for the Mn²⁺-supported reaction. The compound exhibits noncompetitive inhibitory kinetics, indicating that it does not perturb substrate binding. Neither the O-methylated version of the compound nor the unsubstituted imide inhibit substrate cleavage, which is consistent with a specific interaction of the N-hydroxyimide with two closely positioned divalent metal ions. A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism.     

A Divalent Cation Stabilizes the Active Conformation of the B. subtilis RNase P·Pre-tRNA Complex: A Role for an Inner-Sphere Metal Ion in RNase P

Metal ions interact with RNA to enhance folding, stabilize structure, and, in some cases, facilitate catalysis. Assigning functional roles to specifically bound metal ions presents a major challenge in analyzing the catalytic mechanisms of ribozymes. Bacillus subtilis ribonuclease P (RNase P), composed of a catalytically active RNA subunit (PRNA) and a small protein subunit (P protein), catalyzes the 5′-end maturation of precursor tRNAs (pre-tRNAs). Inner-sphere coordination of divalent metal ions to PRNA is essential for catalytic activity but not for the formation of the RNase P·pre-tRNA (enzyme-substrate, ES) complex. Previous studies have demonstrated that this ES complex undergoes an essential conformational change (to the ES^? conformer) before the cleavage step. Here, we show that the ES^? conformer is stabilized by a high-affinity divalent cation capable of inner-sphere coordination, such as Ca(II) or Mg(II). Additionally, a second, lower-affinity Mg(II) activates cleavage catalyzed by RNase P. Structural changes that occur upon binding Ca(II) to the ES complex were determined by time-resolved Förster resonance energy transfer measurements of the distances between donor-acceptor fluorophores introduced at specific locations on the P protein and pre-tRNA 5′ leader. These data demonstrate that the 5′ leader of pre-tRNA moves 4 to 6 Å closer to the PRNA·P protein interface during the ES-to-ES^? transition and suggest that the metal-dependent conformational change reorganizes the bound substrate in the active site to form a catalytically competent ES^? complex.     

Interaction of human DNA polymerase beta with ions of copper, lead, and cadmium

Under appropriate conditions, divalent copper, lead, and cadmium ions significantly inhibit human DNA polymerase β (following accepted convention, the term DNA polymerase β refers to the low-molecular-weight, 3–4 S DNA polymerase of eukaryotic cells) at concentrations below 10^?5m. Each metal showed apparent linear noncompetitive inhibition kinetics with respect to the template primer and the deoxynucleoside triphosphate substrate, indicating that complex formation with these components does not account for the inhibition. Apparently, neither lead nor cadmium inhibit by displacing required zinc atoms from the polymerase. The interaction of the metals with the enzyme can be reversed or prevented by EDTA or by thiol compounds, except that inhibition by cadmium ions can be reversed by monothiols but not by dithiols. The metals probably do not inhibit through reaction with thiol groups since the inhibition is not decreased by pretreating the enzyme with N-ethylmaleimide. Although divalent zinc is moderately inhibitory in manganese activated poly(dT) synthesis on a poly(dA) template, it can fill the requirement for a divalent metal ion and, under the conditions tested, is about 60% as effective as Mn²⁺.