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1.
Magnesium-dependent adenosine triphosphatase, purified from sheep kidney medulla using digitonin, has been characterized in a series of kinetic and magnetic resonance studies. Kinetic studies of divalent metal activation using either Mg2+ or Mn2+ indicate a biphasic response to divalent cations. Apparent Km values of 23 μm for free Mg2+ and 3.3 μm for free Mn2+ are obtained at low levels of added metal, while Km values of 0.50 mm for free Mg2+ and 0.43 mm for free Mn2+ are obtained at much higher levels of divalent cations. In all cases the kinetic data indicate that the binding of divalent metals is independent of the substrate, ATP. Kinetic studies of the substrate requirements of the Mg2+-ATPase also yield biphasic Lineweaver-Burk plots. At low ATP concentrations, kinetic studies yield apparent Km values for free ATP of 6.0 and 1.4 μm with Mg2+ and Mn2+, respectively, as the activating divalent metals. At much higher levels of ATP the response of the enzyme to ATP changes so that Km values for free ATP of 8.0 and 2.0 mm are obtained for Mg2+ and Mn2+, respectively. In both cases, however, the binding of ATP is independent of added metal. ADP inhibits the Mg2+-ATPase and the kinetic data indicate that ADP competes with ATP at both the high and low affinity sites. Dixon plots of the data are consistent with competitive inhibition at both ATP sites, with Ki values of 10.5 μm and 4.5 mm. Electron paramagnetic resonance and water proton relaxation rate studies show that the enzyme binds 1 g ion of Mn2+ per 469,000 g of protein. The Mn2+ binding studies yield a KD for Mn2+ at the single high affinity site of 2 μm, in good agreement with the kinetically determined activator constant for Mn2+ at low Mn2+ levels. Moreover, the EPR binding studies also indicate the existence of 34 weak sites for Mn2+ per single high affinity Mn2+ site. The KD for Mn2+ at these sites is 0.55 mm, in good agreement with the kinetic activator constant for Mn2+ of 0.43 mm, consistent with additional activation of the enzyme by the large number of weaker metal binding sites. The enhancement of water proton relaxation by Mn2+ in the presence of the enzyme is also consistent with the tight binding of a single Mn2+ ion per 469,000 Mr protein and the weaker binding of a large number of divalent metal ions. Analysis of the data yields a value for the enhancement for bound Mn2+ at the single tight site, ?b, of 5 and an enhancement at the 34 weak sites of 11. The frequency dependence of water proton relaxation by Mn2+ at the single tight site yields a dipolar correlation time (constant from 8–60 MHz) of 3.18 × 10?9 s. The kinetics and metal binding studies, together with the effect of temperature on ATPase activity at high and low levels of ATP, are consistent with the existence in this preparation of a single Mg2+-ATPase, with high and low affinity sites for divalent metals and for ATP. Observations of both high and low affinities for ATP have been made with two other purified ATPases. The similarities of these systems to the Mg2+-ATPase described here are discussed. 相似文献
2.
Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5′ kinase, a central 2′,3′ phosphatase and a C-terminal ligase. The phosphatase module is a Mn2+-dependent phosphodiesterase–monoesterase that dephosphorylates 2′,3′-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn2+ and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn2+ in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase. 相似文献
3.
Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis 总被引:1,自引:0,他引:1
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HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg2+ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP–RNA interactions. The best divalent cations were Mn2+, Zn2+ and Cd2+, followed by Mg2+, Co2+ and Ni2+, while Cu2+, Yb2+ and Hg2+ were ineffective. In the HutP–RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein–RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg2+, Mn2+ and Ba2+), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition. 相似文献
4.
Cytosolic fructose-1,6-bisphosphatase from spinach (Spinacia oleracea L.) leaves was purified over 1700-fold. The final preparation was specific for fructose-1,6-bisphosphate in the presence of either Mg2+ or Mn2+, and was free of interfering enzyme activities. Ca2+ was an effector of fructose-1,6-bisphosphatase activity, and showed different kinetics, depending on whether Mg2+ or Mn2+ was used as cofactor. In the presence of 5 millimolar Mg2+, Ca2+ appeared as activator or as inhibitor of the enzyme at low or high levels of substrate, respectively. In both cases, a rise in affinity for fructose-1,6-bisphosphate was observed. A model is proposed to describe the complex interaction of fructose-1,6-bisphosphatase with its substrate and Ca2+. However, with Mn2+ (60 micromolar) as cofactor, Ca2+ exhibited the Michaelis-Menten kinetics of a noncompetitive inhibitor. When assayed at constant substrate concentration, Ca2+ behaves as a competitive or noncompetitive inhibitor, depending on the use of Mg2+ or Mn2+ as cofactor, respectively, with a positive cooperativity in both cases. Fructose-2,6-bisphosphate showed a classic competitive allosteric inhibition in the presence of Mg2+ as cofactor, but this effect was low with Mn2+. From these results we suggest that Ca2+ plays a role in the in vivo regulation of cytosolic fructose-1,6-bisphosphatase. 相似文献
5.
Binding of fructose-6-P and Pi to rabbit liver fructose bisphosphatase has been analyzed in terms of four negatively cooperative binding sites per enzyme tetramer. The association of fructose-6-P occurs in the absence of divalent metal ion, although the extent of binding is increased in the order Mg2+ < Zn2+ < Mn2+. The binding of Pi shows an absolute requirement for divalent metal ion with Mn2+ being more effective than Mg2+. The interaction of the enzyme with the substrate analog, (α + β) methyl-d-fructofuranoside-1,6-P2 in the presence of Mn2+ closely resembles that found for fructose-1,6-P2 in the absence of Mn2+, although the measured constants are on average an order of magnitude smaller. Combination experiments with the three ligands show that the binding follows an identical ordered sequence, i.e., the tighter sites are initially occupied regardless of the ligand's identity. The binding of Pi or fructose-6-P is not altered by the presence of the other. Comparison of binding constant with Ki values obtained from steady-state assays permits identification of the catalytic sites expressed in the latter. The association of Mn2+ at the catalytic site can be induced by fructose-6-P or the substrate analog suggesting that a 1-phosphoryl group enhances but is not necessary for Mn2+ binding at this site. The binding of AMP is decreased in the presence of substrate analog relative to fructose-1,6-P2, suggesting that the 2-hydroxyl serves as a “molecular signal.” From the single and combined binding experiments, a calculation of the equilibrium constant for the overall hydrolysis reaction on the enzyme surface in the presence of Mn2+ has been carried out and an estimate made for the Mg2+ case. 相似文献
6.
George D. Case 《生物化学与生物物理学报:生物膜》1975,375(1):69-86
Measurements of water proton spin relaxation enhancements (ε) can be used to discriminate high-affinity binding of Mn2+ or Gd3+ to biological membranes, from low-affinity binding. In rat liver mitochondria, εb values of approx. 11 are observed upon binding of Mn2+ to the inner membrane, while internal or low-affinity binding remains invisible to this technique. Energy-driven Mn2+ uptake by liver mitochondria results in the subsequent decay of ε1.Comparison of ε1 with the initial velocity of Mn2+ uptake in rat liver mitochondria reveals a linear correlation, which holds at all temperatures between 0 °C and 40 °C, regardless of the mitochondrial protein concentration. Consequently, enhancement appears to reflect the binding of Mn2+ to the divalent cation pump.Binding of Mn2+ to blowfly flight muscle also results in substantial ε1, which is associated with the glycerol-1-phosphate dehydrogenase instead of divalent cation transport. Consequently, no decay in ε1 due to uptake occurs after Mn2+ is bound.Lanthanide ions are also bound and transported by mitochondria. Addition of Gd3+ to pigeon heart or rat liver mitochondria results in εb ≈ 5–6, which decays with similar kinetics in both systems. The uptake velocity of Gd3+ in rat liver mitochondria is about the rate with which Mn2+ is transported. Lanthanides also diminish ε1 due to the addition of Mn2+, and greatly retard the Mn2+ uptake kinetics. The presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone depresses ε1 upon addition of Mn2+ or Gd3+ and also uncouples energy-driven uptake. On the other hand, prolonged anaerobic incubation in the presence of antimycin and rotenone exhausts the mitochondria of their energy stores, blocks the uptake of Mn2+, but does not affect ε1 significantly. Evidently, the uncoupler-induced disappearance of divalent cation binding sites is not the result of “de-energization”.Measurements of ε1 at several NMR frequencies indicate a correlation time (τb) for carrier-bound Mn2+ in rat liver mitochondria between 20 ns and 4 ns as one varies the temperature between 10 °C and 30 °C. The 13 Kcal/mole activation energy for τb suggests that the 11 ns time constant at room temperature represents the movement of the MnII-carrier complex. On the other hand, τb is probably approx. 100 times too short to represent the rotational motion of a carrier protein. Apparently, Mn2+ binds to a small arm of the carrier which moves independently of the main body of any protein.In addition to Mn(H2O)62+, other complexes of Mn2+ may also be bound and transported by rat liver mitochondria. Only a small increase in ε1 occurs upon addition of MnHPO4, yet this species is accumulated by the mitochondria. Consequently, the carrier does not recognize divalent metal ions on the basis of charge. 相似文献
7.
The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His244 or His266 residue. The binding of Zn2+ to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co2+, Ca2+, Ni2+, Mg2+, and Mn2+. The strength of Ca2+, Co2+, Mg2+, Mn2+ and Ni2+ binding to the site close to His244 is stronger than that of these divalent metal ions binding to the site close to His266. 相似文献
8.
Norimichi Nomura Yayoi Morinaga Nobuaki Shirai Yoshihiko Sako 《Nucleic acids research》2005,33(13):e116
Over 50 introns have been reported in archaeal rRNA genes (rDNAs), a subset of which nests putative homing endonuclease (HEase) genes. Here, we report the identification and characterization of a novel archaeal LAGLIDADG-type HEase, I-ApeI, encoded by the ApeK1.S908 intron within the 16S rDNA of Aeropyrum pernix K1. I-ApeI consists of 222 amino acids and harbors two LAGLIDADG-like sequences. It recognizes the 20 bp non-palindromic sequence 5′-GCAAGGCTGAAAC↓TTAAAGG and cleaves target DNA to produce protruding tetranucleotide 3′ ends. Either Mn2+ or Co2+ can be substituted for Mg2+ as a cofactor in the cleavage reaction. Of the 20 bases within the minimal recognition site, 7 are essential for cleavage and are located at positions proximal to the cleavage sites. 相似文献
9.
The extent and modes of binding of the divalent metal ions Mn2+ and Co2+ to DNA and the effects of salt on the binding have been studied by measurements of the effects of these paramagnetic metal ions on the longitudinal and transverse relaxation rates of the protons of the solvent water molecules, a technique that is sensitive to overall binding. The number of water molecules coordinated to the DNA–bound Mn2+ and Co2+ is found to be between five and six, and the electron spin relaxation times and the electron-nuclear hyperfine constants associated with Mn2+ and Co2+ are little or not affected by the binding. These observations indicate little disturbance of the hydration sphere of Mn2+ and Co2+ upon binding to DNA. An average 2–3-fold reduction in the exchange rate of the water of hydration of the bound metal ions and an order-of-magnitude increase in their rotational correlation time are attributed to hydrogen-bond formation with the DNA. The binding constants of Mn2+ to DNA, at metal concentrations approaching zero, are found to be inversely proportional to the second power of the salt concentration, in agreement with the predictions of Manning's polyelectrolyte theory. A remarkable quantitative agreement with the polyelectrolyte theory is also obtained for the anticooperativity in the binding of Mn2+ to DNA, although the experimental results can be well accounted for by another simple electrostatic model. The various modes of binding of divalent metal ions to DNA are discussed. 相似文献
10.
Isobel E. Bowles Emily H. Pool Benjamin S. Lancaster Emily K. Lawson Christopher P. Savas Zach J. Kartje Luke Severinac David H. Cho Mark R. Macbeth R. Jeremy Johnson Geoffrey C. Hoops 《Protein science : a publication of the Protein Society》2021,30(8):1554
Mycobacterium tuberculosis virulence is highly metal‐dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations (Cu2+ ≥ Zn2+ > Ni2+ > Co2+) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for k cat rather than K M. Removal of the artificial N‐terminal 6xHis‐tag did not change the metal‐dependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8 Å and 2.0 Å resolution in the presence and absence of Zn2+ with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metal‐dependent inhibition was traced to multiple chelating residues (H202A/E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals. 相似文献
11.
12.
Characterization of in vitro proton pumping by microsomal vesicles isolated from corn coleoptiles 总被引:12,自引:10,他引:2
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Corn (Zea mays L. cv Golden Cross Bantam) coleoptile microsomal vesicles have been isolated which are capable of ATP-driven H+-transport as measured by [14C]methylamine accumulation and quinacrine fluorescence quenching. Formation of the pH gradient in vitro shows a high specificity for ATP·Mg, is temperature-sensitive, exhibits a pH optimum at 7.5, and is inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Of the divalent cations tested, Mn2+ is almost as effective as Mg2+, while Ca2+ is ineffective. Excess divalent cations, particularly Ca2+, reduces the pH gradient. H+ transport is strongly promoted by anions, especially chloride, while potassium does not affect pump activity. Studies with 36Cl− indicate that ATP-driven H+ transport into the vesicles is associated with chloride uptake. Both carbonyl cyanide-m-chlorophenylhydrazone and the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, inhibit methylamine accumulation and 36Cl− uptake. Proton pumping is also blocked by diethyl stilbestrol and N,N′-dicyclohexylcarbodiimide, but is insensitive to oligomycin and vanadate. These properties of the pump are inconsistent with either a mitochondrial or plasma membrane origin. 相似文献
13.
14.
The effects of divalent cations on photoactivation of the latent water-oxidation system in intact chloroplasts isolated from wheat (Triticum aestivum L.) leaves grown under intermittent flash illumination were investigated by using A23187, an ionophore for divalent cations, and the following results were obtained. (a) Photoactivation in the intact chloroplasts was inhibited by A23187, but was restored on addition of a low concentration of Mn2+ (10 μM). (b) A high concentration of Mn2+ (70 μM) was inhibitory, in contrast, for photoactivation, but the inhibition was restored by the coexistence of a suitable concentration of Ca2+ (5 mM). (c) The Ca2+-dependent restoration was inhibited by a high concentration of Mg2+ or Sr2+, but the inhibition was restored by the coexistence of Ca2+. (d) Kinetic analyses of these competitive effects between divalent cations revealed that: (i) High concentration of Ca2+ inhibits photoactivation in competition with Mn2+. (ii) High concentration of Mn2+ inhibits photoactivation in competition with Ca2+. (iii) High concentration of Mg2+ affects photoactivation by inhibiting Ca2+-dependent restoration in competition with Ca2+. Based on these results, we propose that the latent water-oxidation center has two binding sites, each specific for Mn2+ and Ca2+, and that photoactivation takes place in the center having both Mn2+ and Ca2+ on their respective binding sites. 相似文献
15.
Identification of the divalent metal ion binding domain of myosin regulatory light chains using spin-labelling techniques 总被引:1,自引:0,他引:1
By the criterion of their primary structure myosin regulatory light chains belong to the ‘calcium binding protein’ family and are thought to contain domains related to the E-F hand structure found in parvalbumin. However, the presence of deletions and non-conservative substitutions in the regulatory light chains indicates that, of the four domains apparent in their structure, only the first is competent to bind Ca2+ or other divalent metal ions. Electron paramagnetic resonance studies were performed in an attempt to provide experimental verification of this hypothesis. The approach is based on the finding that the paramagnetic Mn2+ ion substitutes for Ca2+ at the divalent metal ion site and that different regulatory light-chain isotypes contain cysteine residues in different domains which may be spin-labelled with a nitroxide derivative. The electron spin interaction between these two paramagnetic centres is a function of the distance of their separation. Clam (Mercenaria mercenaria) regulatory light chain contains a single cysteine residue located near the first domain and, when spin-labelled, the intensity of the nitroxide signal is reduced by 25% on binding one mole of Mn2+. Rabbit skeletal regulatory light chain contains two cysteine residues located in the third and fourth domains and no (<5%) interaction is observed when Mn2+ binds to spin-labelled derivatives. Qualitatively, these results suggest that domain 1 is the most likely candidate for the Mn2+ binding site. A more quantitative evaluation using the Leigh (1970) theory for the dipolar coupling between rigid-lattice electron spins and various models for the regulatory light chain tertiary structure, including that predicted by Kretsinger &; Barry (1975) for the possibly isologous troponin C structure, substantiates this conclusion. 相似文献
16.
Paul H. Weinreb Sheng Li Sharon X. Gao Tong Liu R. Blake Pepinsky Justin A. Caravella Jun H. Lee Virgil L. Woods Jr. 《The Journal of biological chemistry》2012,287(39):32897-32912
We have applied hydrogen-deuterium exchange mass spectrometry, in conjunction with differential scanning calorimetry and protein stability analysis, to examine solution dynamics of the integrin α1 I domain induced by the binding of divalent cations, full-length type IV collagen, or a function-blocking monoclonal antibody. These studies revealed features of integrin activation and α1I-ligand complexes that were not detected by static crystallographic data. Mg2+ and Mn2+ stabilized α1I but differed in their effects on exchange rates in the αC helix. Ca2+ impacted α1I conformational dynamics without altering its gross thermal stability. Interaction with collagen affected the exchange rates in just one of three metal ion-dependent adhesion site (MIDAS) loops, suggesting that MIDAS loop 2 plays a primary role in mediating ligand binding. Collagen also induced changes consistent with increased unfolding in both the αC and allosteric C-terminal helices of α1I. The antibody AQC2, which binds to α1I in a ligand-mimetic manner, also reduced exchange in MIDAS loop 2 and increased exchange in αC, but it did not impact the C-terminal region. This is the first study to directly demonstrate the conformational changes induced upon binding of an integrin I domain to a full-length collagen ligand, and it demonstrates the utility of the deuterium exchange mass spectrometry method to study the solution dynamics of integrin/ligand and integrin/metal ion interactions. Based on the ligand and metal ion binding data, we propose a model for collagen-binding integrin activation that explains the differing abilities of Mg2+, Mn2+, and Ca2+ to activate I domain-containing integrins. 相似文献
17.
Lisa Zheng Adrina Khemlani Natalie Lorenz Jacelyn M. S. Loh Ries J. Langley Thomas Proft 《The Journal of biological chemistry》2015,290(52):31126-31137
Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5′-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 169 μm and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5′-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg2+, Ca2+, or Mn2+. However, Zn2+ inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner, suggesting a role as an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5′-nucleotidase activity and immune evasion properties. 相似文献
18.
Divalent metal ion effects on a mutant histidinol phosphate phosphatase from Salmonella typhimurium 总被引:1,自引:0,他引:1
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 Km of histidinol phosphate at pH 6.5 from 5.5 mm (without Me2+) to 0.14 mm. Ni2+ and Be2+ increased the Km 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 kimn2+ 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. 相似文献
19.
31P-nmr has been used to investigate the specific interaction of three divalent metal ions, Mg2+, Mn2+, and Co+2, with the phosphate groups of DNA. Mg2+ is found to have no significant effect on any of the 31P-nmr parameters (chemical shift, line-width, T1, T2, and NOE) over a concentration range extending from 20 to 160 mM. The two paramagnetic ions, Mn2+ and Co2+, on the other hand, significantly change the 31P relaxation rates even at very low levels. From an analysis of the paramagnetic contributions to the spin–lattice and spin–spin relaxation rates, the effective internuclear metal–phosphorus distances are found to be 4.5 ± 0.5 and 4.1 ± 0.5 Å for Mn2+ and Co2+, respectively, corresponding to only 15 ± 5% of the total bound Mn2+ and Co2+ being directly coordinated to the phosphate groups (inner-sphere complexes). This result is independent of any assumptions regarding the location of the remaining metal ions which may be bound either as outer-sphere complexes relative to the phosphate groups or elsewhere on the DNA, possibly to the bases. Studies of the temperature effects on the 31P relaxation rates of DNA in the absence and presence of Mn2+ and Co2+ yielded kinetic and thermodynamic parameters which characterize the association and dissociation of the metal ions from the phosphate groups. A two-step model was used in the analysis of the kinetic data. The lifetimes of the inner-sphere complexes are 3 × 10?7 and 1.4 × 10?5 s for Mn2+ and Co2+, respectively. The rates of formation of the inner-sphere complexes with the phosphate are found to be about two orders of magnitude slower than the rate of the exchange of the water of hydration of the metal ions, suggesting that expulsion of water is not the rate-determining step in the formation of the inner-sphere complexes. Competition experiments demonstrate that the binding of Mg2+ ions is 3–4 times weaker than the binding of either Mn2+ or Co2+. Since the contribution from direct phosphate coordination to the total binding strength of these metal ion complexes is small (~15%), the higher binding strength of Mn2+ and Co2+ may be attributed either to base binding or to formation of stronger outer-sphere metal–phosphate complexes. At high levels of divalent metal ions, and when the metal ion concentration exceeds the DNA–phosphate concentration, the fraction of inner-sphere phosphate binding increases. In the presence of very high levels of Mg2+ (e.g., 3.1M), the inner-sphere ? outer-sphere equilibrium is shifted toward ~100% inner-sphere binding. A comparison of our DNA results and previous results obtained with tRNA indicates that tRNA and DNA have very similar divalent metal ion binding properties. A comparison of the present results with the predictions of polyelectrolyte theories is presented. 相似文献
20.
The divalent metal ions Cu2+, Co2+, Mn2+, and Zn2+ form complexes with the fluorescent etheno analogs of the adenine nucleotides. The fluorescence intensity is thereby diminished. The binding strength of the metals to etheno-adenosine triphosphate is higher than to etheno-adenosine di- and monophosphate. The quenching effect of the divalent metal ions can be exploited as a simple routine activity measurement for various kinases and phosphohydrolases. 相似文献