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Mg2+ Ions (mg2+ + ion)
Selected AbstractsSignificant activity of a modified ribozyme with N7-deazaguanine at G10.1: the double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymesGENES TO CELLS, Issue 8 2000Yuka Nakamatsu Background Several reports have appeared recently of experimental evidence for a double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes. In one case, hammerhead ribozyme-mediated cleavage was analysed as a function of the concentration of La3+ ions in the presence of a fixed concentration of Mg2+ ions so that the role of metal ions that are directly involved in the cleavage reaction could be monitored. The resultant bell-shaped curve for activation of cleavage was used to support the proposed double-metal-ion mechanism of catalysis. However, other studies have demonstrated that the binding of a metal ion (the most conserved P9 metal ion) to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and to the N7 of nucleotide G10.1 is critical for efficient catalysis, despite the large distance (,20 Ĺ) between the P9 metal ion and the labile phosphodiester group in the ground state. In fact, it was demonstrated that an added Cd2+ ion binds first to the pro-Rp phosphoryl P9 oxygen but not with the pro-Rp phosphoryl oxygen at the cleavage site. Results In earlier discussions, it was difficult to completely exclude the possibility that La3+ ions might have replaced the P9 metal ion and, as a result, created conditions represented by the bell-shaped curve. In order to clarify this situation, we examined a chemically synthesized hammerhead ribozyme (7-deaza-R34) that included a minimal modification, namely, an N7-deazaguanine residue in place of G10.1. We compared the kinetic properties of this ribozyme with those of the parental ribozyme (R34). Kinetic analysis revealed that, unlike the cases of added Cd2+ ions, the added La3+ ions did not replace the pre-existing P9 metal ion, and that the replacement of N7 by C7 at G10.1 reduced the catalytic activity to a limited extent. This result indicates that the binding of a Mg2+ ion to N7 at G10.1 is catalytically important but not indispensable. Most importantly, 7-deaza-R34 also yielded a bell-shaped curve upon addition of La3+ ions to the reaction mixture. Conclusions Since the data based on our experiments with 7-deaza-R34 are completely free from potential artefacts, due to the binding of a La3+ ion to N7 at G10.1, our results, that 7-deaza-R34 yielded a bell-shaped curve following the addition of La3+ ions to the Mg2+ -background reaction mixture, strongly supports the proposal that a double-metal-ion mechanism is operative in the cleavage reaction which is catalysed by hammerhead ribozymes. [source] Cation-Enhanced Deprotonation of Water by a Strong PhotobaseISRAEL JOURNAL OF CHEMISTRY, Issue 2 2009Noga Munitz We have used picosecond fluorescence spectroscopy to study the proton-dissociation dynamics of bulk water and H2O molecules solvating Mg2+ ions in aqueous solutions. We have analyzed the photo-initiated proton-transfer reaction to a photobase 6-aminoquinoline by the Collins-Kimball approach and have modeled the ensuing bimolecular reaction dynamics by the Smoluchowski equation with radiation boundary conditions. We have found the on-contact proton transfer rate to follow the Marcus free-energy relation for proton transfer and estimate by this rate-equilibrium correlation the considerable enhancement in the acidity of the water molecules solvating the Mg2+ ion. Our findings may be used in the study of metallo-enzymes such as carbonic anhydrases (CAs), which catalyze the reversible addition reaction of OH, to CO2 by increasing the reactivity of the zinc-bound water molecules by means of stabilizing the product of water dissociation, the OH, anion. [source] Elucidation of spermidine interaction with nucleotide ATP by multiple NMR techniquesMAGNETIC RESONANCE IN CHEMISTRY, Issue 2 2010Zhiyan Song Abstract Interaction of polyamines with nucleotides plays a key role in many biological processes. Here we use multiple NMR techniques to characterize interaction of spermidine with adenosine 5,-triphosphate (ATP). Two-dimensional 1H- 15N spectra obtained from gs-HMBC experiments at varied pH show significant shift of N-1 peak around pH 2.0,7.0 range, suggesting that spermidine binds to N-1 site of ATP base. The binding facilitates N-1 deprotonation, shifting its pKa from 4.3 to 3.4. By correlating 15N and 31P chemical shift data, it is clear that spermidine is capable of concurrently binding to ATP base and phosphate sites around pH 4.0,7.0. The self-diffusion constants derived from 1H PFG-diffusion measurements provide evidence that binding of spermidine to ATP is in 1:1 ratio, and pH variations do not induce significant nucleotide self-association in our samples. 31P spectral analysis suggests that at neutral pH, Mg2+ ion competes with spermidine and shows stronger binding to ATP phosphates. From 31P kinetic measurements of myosin-catalyzed ATP hydrolysis, it is found that binding of spermidine affects the stability and reactivity of ATP. These NMR results are important for advancing the studies on nucleotide,polyamine interaction and its impact on nucleotide structures and activities under varied conditions. Copyright © 2009 John Wiley & Sons, Ltd. [source] Spectroscopic investigation of Nd3+ in ASLPHYSICA STATUS SOLIDI (C) - CURRENT TOPICS IN SOLID STATE PHYSICS, Issue 1 2005A. Lupei Abstract Nd3+ -activated strontium lanthanum aluminate Sr1,xNdyLax,yMgxAl12,xO19 (ASL: Nd) crystals are investigated especially for 4F3/2,4I9/2 laser emission in 900 nm range. The optimisation of laser emission characteristics requires a detailed knowledge of spectral features and their dependence on composition. The spectral investigation of Nd3+ in ASL on an extended composition range (especially small x) reveals new spectral features. Connecting spectral and structural data, the role of charge compensator Mg2+ ion in the structure of the two main Nd3+ structural centers in ASL, C1 and C2, is proposed. It is inferred that these results enable a rigorous selection of the optimal composition for improving laser properties. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Structures of human deoxycytidine kinase product complexesACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2007Erika V. Soriano Human deoxycytidine kinase (dCK) is involved in the nucleotide-biosynthesis salvage pathway and has also been shown to phosphorylate several antitumor and antiviral prodrugs. The structures of dCK alone and the dead-end complex of dCK with substrate nucleoside and product ADP or UDP have previously been reported; however, there is currently no structure available for a substrate or product complex. Here, the structures of dCK complexes with the products dCMP, UDP and Mg2+ ion, and with dAMP, UDP and Mg2+ ion are reported. Structural comparisons show that the product complexes with UDP and a dead-end complex with substrate and UDP have similar active-site conformations. [source] Crystallization and preliminary X-ray analysis of a DNA dodecamer containing 2,-deoxy-5-formyluridine; what is the role of magnesium cation in crystallization of Dickerson-type DNA dodecamers?ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2001Masaru Tsunoda To investigate the role of divalent cations in crystal packing, four different crystals of a Dickerson-type dodecamer with the sequence d(CGCGAATXCGCG), containing 2,-deoxy-5-formyluridine at X, were obtained under several conditions with and without divalent cations. The crystal structures are all isomorphous. The octahedrally hydrated magnesium cations found in the major groove cement the two neighbouring duplexes along the b axis. In the Mg2+ -free crystals, a five-membered ring of water molecules occupies the same position as the magnesium site and connects the two duplexes similarly to the hydrated Mg2+ ion. It has been concluded that water molecules can take the place of the hydrated magnesium cation in crystallization, but the magnesium cation is more effective and gives X-ray diffraction at slightly higher resolution. In all four crystals, the 5-formyluracil residues form the canonical Watson,Crick pair with adenine residues. [source] Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTPBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005Jie Bao Abstract The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use. © 2005 Wiley Periodicals, Inc. [source] Oxygen binding and its allosteric control in hemoglobin of the primitive branchiopod crustacean Triops cancriformisFEBS JOURNAL, Issue 13 2007Ralph Pirow Branchiopod crustaceans are endowed with extracellular, high-molecular-mass hemoglobins (Hbs), the functional and allosteric properties of which have largely remained obscure. The Hb of the phylogenetically ancient Triops cancriformis (Notostraca) revealed moderate oxygen affinity, cooperativity and pH dependence (Bohr effect) coefficients: P50 = 13.3 mmHg, n50 = 2.3, and , = ,0.18, at 20 °C and pH 7.44 in Tris buffer. The in vivo hemolymph pH was 7.52. Bivalent cations increased oxygen affinity, Mg2+ exerting a greater effect than Ca2+. Analysis of cooperative oxygen binding in terms of the nested Monod,Wyman,Changeux (MWC) model revealed an allosteric unit of four oxygen-binding sites and functional coupling of two to three allosteric units. The predicted 2 × 4 and 3 × 4 nested structures are in accord with stoichiometric models of the quarternary structure. The allosteric control mechanism of protons comprises a left shift of the upper asymptote of extended Hill plots which is ascribable to the displacement of the equilibrium between (at least) two high-affinity (relaxed) states, similar to that found in extracellular annelid and pulmonate molluscan Hbs. Remarkably, Mg2+ ions increased oxygen affinity solely by displacing the equilibrium between the tense and relaxed conformations towards the relaxed states, which accords with the original MWC concept, but appears to be unique among Hbs. This effect is distinctly different from those of ionic effectors (bivalent cations, protons and organic phosphates) on annelid, pulmonate and vertebrate Hbs, which involve changes in the oxygen affinity of the tense and/or relaxed conformations. [source] Stage-specific expression of Caenorhabditis elegans ribonuclease H1 enzymes with different substrate specificities and bivalent cation requirementsFEBS JOURNAL, Issue 2 2006Hiromi Kochiwa Ribonuclease H1 (RNase H1) is a widespread enzyme found in a range of organisms from viruses to humans. It is capable of degrading the RNA moiety of DNA,RNA hybrids and requires a bivalent ion for activity. In contrast with most eukaryotes, which have one gene encoding RNase H1, the activity of which depends on Mg2+ ions, Caenorhabditis elegans has four RNase H1-related genes, and one of them has an isoform produced by alternative splicing. However, little is known about the enzymatic features of the proteins encoded by these genes. To determine the differences between these enzymes, we compared the expression patterns of each RNase H1-related gene throughout the development of the nematode and the RNase H activities of their recombinant proteins. We found gene-specific expression patterns and different enzymatic features. In particular, besides the enzyme that displays the highest activity in the presence of Mg2+ ions, C. elegans has another enzyme that shows preference for Mn2+ ion as a cofactor. We characterized this Mn2+ -dependent RNase H1 for the first time in eukaryotes. These results suggest that there are at least two types of RNase H1 in C. elegans depending on the developmental stage of the organism. [source] Molecular dynamics simulations of solvated UDP,glucose in interaction with Mg2+ cationsFEBS JOURNAL, Issue 20 2001Pavla Petrová ,Glycosyltransferases are key enzymes involved in biosynthesis of oligosaccharides. Nucleotide-sugars, the glycosyltransferase substrates, serve as activated donors of sugar residues during the enzymatic reaction Although very little is known about the catalytic mechanism of these enzymes, it appears that the catalytic activity in most glycosyltransferases is dependent upon the presence of a divalent cation, for example Mn2+ or Mg2+. It is not known whether the ion is bound to the enzyme before its interaction with the substrate, or if it binds the substrate before the enzymatic reaction to modify its conformation to fit better the active site of the enzyme. We have inspected the latter possibility by running four 2-ns molecular dynamics trajectories on fully solvated UDP-glucose in the presence of Mg2+ ions. Our results indicate that the divalent cation interacts strongly with the nucleotide-sugar in solution, and that it can alter its conformational behavior. It is also shown that a conformation of the pyrophosphate moiety that results in an eclipsed or almost eclipsed orientation of two of the oxygen atoms, and which is found in protein interacting with a nucleotide di- or tri-phosphate X-ray data, is energetically favored. The results are also discussed in light of existing NMR data, and are found to be in a good agreement with them. [source] Significant activity of a modified ribozyme with N7-deazaguanine at G10.1: the double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymesGENES TO CELLS, Issue 8 2000Yuka Nakamatsu Background Several reports have appeared recently of experimental evidence for a double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes. In one case, hammerhead ribozyme-mediated cleavage was analysed as a function of the concentration of La3+ ions in the presence of a fixed concentration of Mg2+ ions so that the role of metal ions that are directly involved in the cleavage reaction could be monitored. The resultant bell-shaped curve for activation of cleavage was used to support the proposed double-metal-ion mechanism of catalysis. However, other studies have demonstrated that the binding of a metal ion (the most conserved P9 metal ion) to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and to the N7 of nucleotide G10.1 is critical for efficient catalysis, despite the large distance (,20 Ĺ) between the P9 metal ion and the labile phosphodiester group in the ground state. In fact, it was demonstrated that an added Cd2+ ion binds first to the pro-Rp phosphoryl P9 oxygen but not with the pro-Rp phosphoryl oxygen at the cleavage site. Results In earlier discussions, it was difficult to completely exclude the possibility that La3+ ions might have replaced the P9 metal ion and, as a result, created conditions represented by the bell-shaped curve. In order to clarify this situation, we examined a chemically synthesized hammerhead ribozyme (7-deaza-R34) that included a minimal modification, namely, an N7-deazaguanine residue in place of G10.1. We compared the kinetic properties of this ribozyme with those of the parental ribozyme (R34). Kinetic analysis revealed that, unlike the cases of added Cd2+ ions, the added La3+ ions did not replace the pre-existing P9 metal ion, and that the replacement of N7 by C7 at G10.1 reduced the catalytic activity to a limited extent. This result indicates that the binding of a Mg2+ ion to N7 at G10.1 is catalytically important but not indispensable. Most importantly, 7-deaza-R34 also yielded a bell-shaped curve upon addition of La3+ ions to the reaction mixture. Conclusions Since the data based on our experiments with 7-deaza-R34 are completely free from potential artefacts, due to the binding of a La3+ ion to N7 at G10.1, our results, that 7-deaza-R34 yielded a bell-shaped curve following the addition of La3+ ions to the Mg2+ -background reaction mixture, strongly supports the proposal that a double-metal-ion mechanism is operative in the cleavage reaction which is catalysed by hammerhead ribozymes. [source] Kinetics of Calcium Carbonate (CaCO3) Precipitation from a Icel-Yavca Dolomite Leach Solution by a Gas (Carbon Dioxide)/Liquid ReactionHELVETICA CHIMICA ACTA, Issue 3 2009Mehmet Yildirim Abstract The effects of time, CO2 -gas-injection pressure, and bulk temperature on the precipitation of Ca2+ ions as a precipitated calcium carbonate (PCC) from a dolomite leach solution were investigated. Precipitation periods from 1 to 7,min were examined, and experiments were run at CO2 -injection pressures of 200,800,kPa. Effects of bulk temperature were studied in the range from 40 to 70°, and precipitation rates of PCC were determined by measuring the Ca2+ concentrations in the initial and effluent solutions. Influences of these parameters on the subsequent incorporation of Mg2+ ions with the precipitate are discussed in detail. Kinetic analysis of the precipitation was performed by considering the rates as a function of CO -ion concentrations. Results obtained by this process clearly show that Ca2+ ions in the solution can successfully be precipitated as a calcium carbonate product containing 54.70% of CaO and 0.77% MgO, at the rate of 2.01,mM h,1. [source] The NMDA receptor antagonist memantine as a symptomatological and neuroprotective treatment for Alzheimer's disease: preclinical evidenceINTERNATIONAL JOURNAL OF GERIATRIC PSYCHIATRY, Issue S1 2003Wojciech Danysz Abstract There is increasing evidence for the involvement of glutamate-mediated neurotoxicity in the pathogenesis of Alzheimer's disease (AD). We suggest that glutamate receptors of the N-methyl-D-aspartate (NMDA) type are overactivated in a tonic rather than a phasic manner in this disorder. This continuous mild activation may lead to neuronal damage and impairment of synaptic plasticity (learning). It is likely that under such conditions Mg2+ ions, which block NMDA receptors under normal resting conditions, can no longer do so. We found that overactivation of NMDA receptors using a direct agonist or a decrease in Mg2+ concentration produced deficits in synaptic plasticity (in vivo: passive avoidance test and/or in vitro: LTP in the CA1 region). In both cases, memantine,an uncompetitive NMDA receptor antagonists with features of an ,improved' Mg2+ (voltage-dependency, kinetics, affinity),attenuated this deficit. Synaptic plasticity was restored by therapeutically-relevant concentrations of memantine (1,,M). Moreover, doses leading to similar brain/serum levels provided neuroprotection in animal models relevant for neurodegeneration in AD such as neurotoxicity produced by inflammation in the NBM or ,-amyloid injection to the hippocampus. As such, if overactivation of NMDA receptors is present in AD, memantine would be expected to improve both symptoms (cognition) and to slow down disease progression because it takes over the physiological function of magnesium. Copyright © 2003 John Wiley & Sons, Ltd. [source] Cation-Enhanced Deprotonation of Water by a Strong PhotobaseISRAEL JOURNAL OF CHEMISTRY, Issue 2 2009Noga Munitz We have used picosecond fluorescence spectroscopy to study the proton-dissociation dynamics of bulk water and H2O molecules solvating Mg2+ ions in aqueous solutions. We have analyzed the photo-initiated proton-transfer reaction to a photobase 6-aminoquinoline by the Collins-Kimball approach and have modeled the ensuing bimolecular reaction dynamics by the Smoluchowski equation with radiation boundary conditions. We have found the on-contact proton transfer rate to follow the Marcus free-energy relation for proton transfer and estimate by this rate-equilibrium correlation the considerable enhancement in the acidity of the water molecules solvating the Mg2+ ion. Our findings may be used in the study of metallo-enzymes such as carbonic anhydrases (CAs), which catalyze the reversible addition reaction of OH, to CO2 by increasing the reactivity of the zinc-bound water molecules by means of stabilizing the product of water dissociation, the OH, anion. [source] Raman microspectroscopic study on low-pH-induced DNA structural transitions in the presence of magnesium ionsJOURNAL OF RAMAN SPECTROSCOPY, Issue 10 2002C. M. Muntean Low-pH-induced DNA structural changes were investigated in the pH range 6.8,2.10 by Raman microspectroscopy. Measurements were carried out on calf thymus DNA in the presence of low concentrations of Mg2+ ions. Vibrational spectra are presented in the wavenumber region 500,1650 cm,1. Large changes in the Raman spectra of calf-thymus DNA were observed on lowering the pH value. These are due to protonation and unstacking of the DNA bases during DNA melting and also to changes in the DNA backbone conformation. The intensities of the Raman bands of guanine (681 cm,1), adenine (728 cm,1), thymine (752 cm,1) and cytosine (785 cm,1), typical of the C2,- endo - anti conformation of B-DNA, are discussed. The B-form marker near 835 cm,1 and the base vibrations in the higher wavenumber region (1200,1680 cm,1) are analysed. Effects of low pH value upon the protonation mechanism of opening AT and changing the protonation of GC base pairs in DNA are discussed. Copyright © 2002 John Wiley & Sons, Ltd. [source] Novel Coagulation Method for Direct Coagulation Casting of Aqueous Alumina Slurries Prepared Using a Poly(Acrylate) DispersantJOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 2 2008Kuttan Prabhakaran Coagulation of concentrated aqueous alumina slurries prepared using an ammonium poly(acrylate) dispersant by MgO has been studied for direct coagulation casting (DCC). A small amount of MgO (0.2 wt% of alumina) increased the viscosity of the concentrated alumina slurry with time and finally transformed it into a stiff gel. The mechanism of coagulation is proposed such that the time-delayed in situ generation of Mg2+ ions from the sparingly soluble MgO forms Mg,poly(acrylate) with the unadsorbed ammonium poly(acrylate) molecules in solution that shift the poly(acrylate) adsorption equilibrium toward the left by depleting the poly(acrylate) molecules adsorbed on the alumina particle surface. This leads to insufficient dispersant coverage on the particle surface and coagulation of the slurry. DCC using MgO is possible only if the slurry is prepared at a dispersant concentration higher than that required for optimum dispersion as the slurries prepared at the optimum dispersant concentration underwent premature coagulation. The gelation time could be tailored within 20 min to a few hours by maintaining the temperature in the range of 70°,30°C. The wet coagulated bodies prepared from 50 vol% alumina slurry showed a compressive strength of nearly 0.05 MPa. [source] The solution structure of the Mg2+ form of soybean calmodulin isoform 4 reveals unique features of plant calmodulins in resting cellsPROTEIN SCIENCE, Issue 3 2010Hao Huang Abstract Soybean calmodulin isoform 4 (sCaM4) is a plant calcium-binding protein, regulating cellular responses to the second messenger Ca2+. We have found that the metal ion free (apo-) form of sCaM4 possesses a half unfolded structure, with the N-terminal domain unfolded and the C-terminal domain folded. This result was unexpected as the apo-forms of both soybean calmodulin isoform 1 (sCaM1) and mammalian CaM (mCaM) are fully folded. Because of the fact that free Mg2+ ions are always present at high concentrations in cells (0.5,2 mM), we suggest that Mg2+ should be bound to sCaM4 in nonactivated cells. CD studies revealed that in the presence of Mg2+ the initially unfolded N-terminal domain of sCaM4 folds into an ,-helix-rich structure, similar to the Ca2+ form. We have used the NMR backbone residual dipolar coupling restraints 1DNH, 1DC,H,, and 1DC,C, to determine the solution structure of the N-terminal domain of Mg2+ -sCaM4 (Mg2+ -sCaM4-NT). Compared with the known structure of Ca2+ -sCaM4, the structure of the Mg2+ -sCaM4-NT does not fully open the hydrophobic pocket, which was further confirmed by the use of the fluorescent probe ANS. Tryptophan fluorescence experiments were used to study the interactions between Mg2+ -sCaM4 and CaM-binding peptides derived from smooth muscle myosin light chain kinase and plant glutamate decarboxylase. These results suggest that Mg2+ -sCaM4 does not bind to Ca2+ -CaM target peptides and therefore is functionally similar to apo-mCaM. The Mg2+ - and apo-structures of the sCaM4-NT provide unique insights into the structure and function of some plant calmodulins in resting cells. [source] Mobile loop mutations in an archaeal inositol monophosphatase: Modulating three-metal ion assisted catalysis and lithium inhibitionPROTEIN SCIENCE, Issue 2 2010Zheng Li Abstract The inositol monophosphatase (IMPase) enzyme from the hyperthermophilic archaeon Methanocaldococcus jannaschii requires Mg2+ for activity and binds three to four ions tightly in the absence of ligands: KD = 0.8 ,M for one ion with a KD of 38 ,M for the other Mg2+ ions. However, the enzyme requires 5,10 mM Mg2+ for optimum catalysis, suggesting substrate alters the metal ion affinity. In crystal structures of this archaeal IMPase with products, one of the three metal ions is coordinated by only one protein contact, Asp38. The importance of this and three other acidic residues in a mobile loop that approaches the active site was probed with mutational studies. Only D38A exhibited an increased kinetic KD for Mg2+; D26A, E39A, and E41A showed no significant change in the Mg2+ requirement for optimal activity. D38A also showed an increased Km, but little effect on kcat. This behavior is consistent with this side chain coordinating the third metal ion in the substrate complex, but with sufficient flexibility in the loop such that other acidic residues could position the Mg2+ in the active site in the absence of Asp38. While lithium ion inhibition of the archaeal IMPase is very poor (IC50,250 mM), the D38A enzyme has a dramatically enhanced sensitivity to Li+ with an IC50 of 12 mM. These results constitute additional evidence for three metal ion assisted catalysis with substrate and product binding reducing affinity of the third necessary metal ion. They also suggest a specific mode of action for lithium inhibition in the IMPase superfamily. [source] Structures of vaccinia virus dUTPase and its nucleotide complexesACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2007Alexandra Samal Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate in the presence of Mg2+ ions. The enzyme plays multiple cellular roles by maintaining a low dUTP:dTTP ratio and by synthesizing the substrate for thymidylate synthase in the biosynthesis of dTTP. Although dUTPase is an essential enzyme and has been established as a valid target for drug design, the high degree of homology of vaccinia virus dUTPase to the human enzyme makes the identification of selective inhibitors difficult. The crystal structure of vaccinia virus dUTPase has been solved and the active site has been mapped by crystallographic analysis of the apo enzyme and of complexes with the substrate-analog dUMPNPP, with the product dUMP and with dUDP, which acts as an inhibitor. Analyses of these structures reveal subtle differences between the viral and human enzymes. In particular, the much larger size of the central channel at the trimer interface suggests new possibilities for structure-based drug design. Vaccinia virus is a prototype of the poxviruses. [source] An experiment illustrating the effect of saline stress and ions on the malate dehydrogenase activity in vegetal tissues,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 6 2002Sergio González-Moreno Abstract This paper describes a simple, rapid, and economic protocol to evaluate the effect of saline stress and Na+, K+, Ca2+, and Mg2+ ions on malate dehydrogenase activity in crude extracts from the leaves and roots of Vigna radiata. The main objectives of this work are to illustrate the effect of an environmental stress factor, as well as the in vitro effect of some ions on an enzymatic activity, while introducing the students to the use of Michaelis-Menten kinetics and a graphic procedure to calculate kinetics constants. [source] Assessment of adenyl residue reactivity within model nucleic acids by surface enhanced Raman spectroscopyBIOPOLYMERS, Issue 1 2006Lydie Grajcar Abstract We rank the reactivity of the adenyl residues (A) of model DNA and RNA molecules with electropositive subnano size [Ag] sites as a function of nucleic acid primary sequences and secondary structures and in the presence of biological amounts of Cl, and Na+ or Mg2+ ions. In these conditions A is markedly more reactive than any other nucleic acid bases. A reactivity is higher in ribo (r) than in deoxyribo (d) species [pA > pdA and (pA)n , (pdA)n]. Base pairing decreases A reactivity in corresponding duplexes but much less in r than in d. In linear single and paired dCAG or dGAC loci, base stacking inhibits A reactivity even if A is bulged or mispaired (A.A). dA tracts are highly reactive only when dilution prevents self-association and duplex structures. In d hairpins the solvent-exposed A residues are reactive in CAG and GAC triloops and even more in ATC loops. Among the eight rG1N2R3A4 loops, those bearing a single A (A4) are the least reactive. The solvent-exposed A2 is reactive, but synergistic structural transitions make the initially stacked A residues of any rGNAA loop much more reactive. Mg2+ cross-bridging single strands via phosphates may screen A reactivity. In contrast d duplexes cross-bridging enables "A flipping" much more in rA.U pairs than in dA.T. Mg2+ promotes A reactivity in unpaired strands. For hairpins Mg2+ binding stabilizes the stems, but according to A position in the loops, A reactivity may be abolished, reduced, or enhanced. It is emphasized that not only accessibility but also local flexibility, concerted docking, and cation and anion binding control A reactivity. © 2006 Wiley Periodicals, Inc. Biopolymers 82: 6,28, 2006 This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] ,-Galactosidase from Lactobacillus pentosus: Purification, characterization and formation of galacto-oligosaccharidesBIOTECHNOLOGY JOURNAL, Issue 8 2010Thomas Maischberger Abstract A novel heterodimeric ,-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 UoNPG/mg protein. The Km, kcat and kcat/Km values for lactose and o -nitrophenyl-,-D-galactopyranoside (oNPG) were 38 mM, 20 s -1, 530 M -1·s -1 and 1.67 mM, 540 s -1, 325 000 M -1·s -1, respectively. The temperature optimum of ,-galactosidase activity was 60,65°C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal ,-galactosidases. Mg2+ ions enhanced both activity and stability significantly. L. pentosus ,-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of ,-(1,3) and ,-(1,6) linkages, and the main transgalactosylation products identified were the disaccharides ,-D-Galp -(1,6)- D -Glc, ,-D-Galp -(1,3)- D -Glc, ,- D -Galp -(1,6)- D -Gal, ,- D -Galp -(1,3)- D -Gal, and the trisaccharides ,- D -Galp -(1,3)- D -Lac, ,- D -Galp -(1,6)- D -Lac. [source] Structure of mouse ADP-ribosylhydrolase 3 (mARH3)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2008Christoph Mueller-Dieckmann ADP-ribosylation is a reversible and covalent post-translational modification in which the attachment of ADP-ribose is catalyzed by ADP-ribosyltransferases and the removal of ADP-ribose is catalyzed by ADP-ribosylhydrolases. ADP-ribosylhydrolase 3 from mouse, consisting of 347 amino-acid residues, has been cloned, purified and crystallized. The three-dimensional structure has been resolved at a resolution of 1.8,Ĺ. The structure constitutes a compact all-,-helical protein with two Mg2+ ions located in the active-site crevice. A structural comparison of mouse ADP-ribosylhydrolase 3 with its human orthologue shows a high degree of structural similarity. Furthermore, four prokaryotic proteins deposited in the PDB could be identified as being structurally related. [source] Carbon Dioxide Activation by Surface Excess Electrons: An EPR Study of the CO2, Radical Ion Adsorbed on the Surface of MgOCHEMISTRY - A EUROPEAN JOURNAL, Issue 4 2007Mario Chiesa Dr. Abstract The CO2, radical anion has been generated at the surface of MgO by direct electron transfer from surface trapped excess electrons and characterized by electron paramagnetic resonance spectroscopy. Both 13C and 17O hyperfine structures have been resolved for the first time, leading to a detailed mapping of the unpaired electron spin density distribution over the entire radical anion. The magnetic equivalence of the two O nuclei has been ascertained allowing a side-on adsorption structure at low-coordinate Mg2+ ions to be proposed for the surface stabilized radical. [source] | ||||||||||||||||||||||||||||