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1.
Kojiro Takahashi Henry Weiner James H.J. Hu 《Archives of biochemistry and biophysics》1980,205(2):571-578
The V of horse liver aldehyde dehydrogenase is enhanced twofold in the presence of 0.5 mm Mg2+ ions when assayed in the dehydrogenase reaction. The mechanism of this activation appears to be related to the fact the enzyme changes from functioning with half-of-the-sites reactivity to functioning with all-of-the-sites reactivity. That is, the presteady-state burst magnitude increases from 2 mol NADH formed per mole of tetrameric enzyme to 4 mol formed per mole (K. Takahashi and H. Weiner, J. Biol. Chem., 1980, 255, 8206–8209). Whether this twofold enhancement correlates, in fact, to a change from half-of-the-sites to all-of-the-sites reactivity of the enzyme by Mg2+ ions was investigated by determining the Stoichiometry of coenzyme binding by fluorescence quenching and enhancement methods in the absence and presence of the metal ions. The biphasic Scatchard plots for NAD binding to the enzyme were similar in the absence and presence of Mg2+ ions, while that of NADH binding was monophasic (-Mg2+) and biphasic (+Mg2+). In the presence of p-methoxyacetophenone, a competitive inhibitor for substrate, the stoichiometric titration of coenzyme binding to the ternary complexes (enzyme-NAD(H)-inhibitor) revealed that only 2 mol of NAD or NADH bind in the absence of Mg2+ ions but 4 bind per mole of tetrameric enzyme in the presence of added metal. The fluorescence intensity of NAD's fluorescent derivative, 1,N6-ethenoadenine dinucleotide, bound to the enzyme was also doubled by the addition of Mg2+ ions.The combined binding data show that the stoichiometry of coenzyme binding to aldehyde dehydrogenase in the ternary complex increases from 2 to 4 mol binding per mole of tetrameric enzyme with the addition of Mg2+ ions. This increase in stoichiometry corresponds to the observed changes of burst magnitude obtained from the presteady-state and V in the steady-state kinetics assays. From both results of the kinetics and stoichiometry, we show that horse liver aldehyde dehydrogenase exhibits half-of-the-sites reactivity when in the tetrameric state in the absence of Mg2+ ions, and all-of-the-sites reactivity in the dimeric state in the presence of the metal. 相似文献
2.
Bengt C. Magnusson 《Histochemistry and cell biology》1974,42(3):211-219
Summary The hydrolysis of ATP, AMP and glycerophosphate (GP) at alkaline pH in mineralizing bone and teeth of young mice has been studied histochemically. The substrates were visibly hydrolyzed to the same degree in osteoblasts, cells of stratum intermedium, odontoblasts and subodontoblasts at Ca2+ concentrations ranging from 10 mM to 600 mM. In the ameloblasts, however, only ATP was hydrolyzed. The ATPase activities gradually decreased at increasing Mg2+/Ca2+ ratios. The AMPase and GPase activities, on the other hand, were visibly unaffected. Marked cellular staining, including the nuclei was seen with AMP and GP as substrates when only Mg2+ ions were added. No ATPase activity at all could be recorded in media containing Mg2+ but no Ca2+ ions. The different phosphatase activities in cells involved in hard tissue formation were identically affected by preincubations with solutions containing various concentrations of Ca2+ or Mg2+ ions. The ATPase activity in striated muscle fibres and blood vessel walls, however, was affected differently by the same procedure.The results indicate that the phosphatase activities recorded in osteoblasts, cells of stratum intermedium, odontoblasts and subodontoblasts at alkaline pH belong to one single enzyme. The results also imply that CaATP is the preferred substrate in the enzymatic hydrolysis of ATP in hard-tissue-forming cells. 相似文献
3.
A through study of initial-rate data has been made on carbamoyl phosphate synthetase from bovine liver. On the basis of the results the order of substrate binding to the enzyme is ATPMg followed by HCO3−, ATPMg and NH4+. A model for the enzymic mechanism is proposed, and the rate equations describing it are presented. Details of the derivation of the initial-rate equation for the kinetic mechanism proposed have been deposited as Supplementary Publication SUP 50032 (6 pages) at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7QB, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5. 相似文献
4.
35Cl? quadrupole relaxation was measured in the presence of metal-free alkaline phosphatase and in the presence of Zn2+-alkaline phosphatase. The relaxation data show that for an enzyme containing the minimum amount of zinc needed for full activity—2 g atoms of zinc per mole of protein—there appears to be no binding of halide ions to the protein-bound zinc ions. In contrast, when there is a high metal-enzyme ratio, a large relaxation enhancement is observed, demonstrating coordination of halide ions to the metal ions.Addition of inorganic phosphate causes no change in the 35Cl? relaxation in the presence of metal-free enzyme. However, marked decreases in relaxation are observed upon addition of phosphate to the Zn2+-alkaline phosphatase. The relaxation measurements carried out in the presence of phosphate show that substrate binding does prove to be metal-ion dependent. Furthermore, experiments with inorganic phosphate suggest the tight binding of one phosphate to the alkaline phosphatase. 相似文献
5.
The alkaline phosphatase (EC 3.1.3.1.) from Rhizobium leguminosarum WU235 has been purified. The enzyme is a non-specific phosphomonoesterase, has a molecular weight of 78,500 and a sub-unit molecular weight of 39,400. Magnesium and zinc ions are implicated in the structure of the enzyme; atomic absorption analysis gave 1.9 g-atoms Mg2+ and 1.9–5.1 g-atoms Zn2+ per mole of enzyme. In addition high concentrations of Mg2+ markedly stimulate the enzyme. The phosphatase is inhibited by Li+ and Na+ and stimulated by K+, Rb+ and Cs+, which suggests that the enzyme is K+ activated. 相似文献
6.
Tran TT Hashim SO Gaber Y Mamo G Mattiasson B Hatti-Kaul R 《Journal of inorganic biochemistry》2011,105(7):1000-1007
Phytate, the major source of phosphorus in seeds, exists as a complex with different metal ions. Alkaline phytases are known to dephosphorylate phytate complexed with calcium ions in contrast to acid phytases that act only on phytic acid. A recombinant alkaline phytase from Bacillus sp. MD2 has been purified and characterized with respect to the effect of divalent metal ions on the enzyme activity and stability. The presence of Ca2+ on both the enzyme and the substrate is required for optimal activity and stability. Replacing Ca2+ with Ba2+, Mn2+, Mg2+ and Sr2+ in the phytase resulted in the expression of > 90% of the maximal activity with calcium-phytate as the substrate, while Fe2+ and Zn2+ rendered the enzyme inactive. On the other hand, the calcium loaded phytase showed significant activity (60%) with sodium phytate and lower activity (17-20%) with phytate complexed with only Mg2+, Sn2+ and Sr2+, respectively. On replacing Ca2+ on both the enzyme and the substrate with other metal ions, about 20% of the maximal phytase activity was obtained only with Mg2+ and Sr2+, respectively. Only Ca2+ resulted in a marked increase in the melting temperature (Tm) of the enzyme by 12-21 °C, while Ba2+, Mn2+, Sr2+ or Cu2+ resulted in a modest (2-3.5 °C) increase in Tm. In the presence of 1-5 mM Ca2+, the optimum temperature of the phytase activity was increased from 40 °C to 70 °C, while optimum pH of the enzyme shifted by 0.4-1 pH unit towards the acidic region. 相似文献
7.
In light of the critical role of divalent metal ions in the chemistry of coenzyme NADH analogs, complexation of 1-benzyl-3-substituted(X)-1,4-dihydropyridines (1, X=CONH2; 2, X=CSNH2; 3, X=COOCH3; 4, X=COCH3) with divalent metal ions (Mg2+, Zn2+, and Co2+) in dry acetonitrile was studied spectroscopically and kinetically. Presence of the metal ions causes red-shift of absorption band of NADH analogs and the rate retardation for the reaction between NADH analogs and N-methylacridinium ion. Analysis of the spectroscopic and kinetic data indicates that the NADH analogs form 1 : 1 complexes with the metal ions. The decreasing order of the magnitude of the association constants, K, is 1 2 4 3 for a given metal ion, and Mg2+ Zn2+ > Co2+ for a given NADH analog. The results strongly suggest that the primary binding site for the metal ions is the carbonyl oxygen (or thiocarbonyl sulfur) of the 3-substituent and that the amide nitrogen atom of the 3-substituent of 1 and 2 also ligates the metal ions, forming a bidentate structure and providing extra stability to the complexes of 1 and 2. Inhibition of reaction between NADH analogs and N-methylacridinium ion by the metal ions is attributed to inaccessibility of N-methylacridinium ion to the NADH analogs complexed with metal ions due to electrostatic repulsion. 相似文献
8.
Cheryl L. Wojciechowski Evan R. Kantrowitz 《Biochimica et Biophysica Acta - Proteins and Proteomics》2003,1649(1):68-73
Four independent mutations were introduced to the Escherichia coli alkaline phosphatase active site, and the resulting enzymes characterized to study the effects of Glu as a metal ligand. The mutations D51E and D153E were created to study the effects of lengthening the carboxyl group by one methylene unit at the metal interaction site. The D51E enzyme had drastically reduced activity and lost one zinc per active site, demonstrating importance of the position of Asp51. The D153E enzyme had an increased kcat in the presence of high concentrations of Mg2+, along with a decreased Mg2+ affinity as compared to the wild-type enzyme. The H331E and H412E enzymes were created to probe the requirement for a nitrogen-containing metal ligand at the Zn1 site. The H331E enzyme had greatly decreased activity, and lost one zinc per active site. In the absence of high concentrations of Zn2+, dephosphorylation occurs at an extremely reduced rate for the H412E enzyme, and like the H331E enzyme, metal affinity is reduced. Except at the 153 position, Glu is not an acceptable metal chelating amino acid at these positions in the E. coli alkaline phosphatase active site. 相似文献
9.
Isocitrate dehydrogenase kinase/phosphatase (AceK) is a bifunctional enzyme with both kinase and phosphatase activities that are activated by Mg2+. We have studied the interactions of Mn2+and Mg2+ with AceK using isothermal titration calorimetry (ITC) combined with molecular docking simulations and show for the first time that Mn2+ also activates the enzyme activities. However, Mn2+ and Mg2+ exert their effects by different mechanisms. Although they have similar binding constants (of 1.11?×?105 and 0.98?×?105 M?1, respectively) for AceK and induce conformational changes of the enzyme, they do not compete for the same binding site. Instead Mn2+ appears to bind to the regulatory domain of AceK, and its effect is transmitted to the active site of the enzyme by the conformational change that it induces. The information in this study should be very useful for understanding the molecular mechanism underlying the interaction between AceK and metal ions, especially Mn2+ and Mg2+. 相似文献
10.
The effect of Mg2+ on the thermal inactivation and unfolding of calf intestinal alkaline phosphatase has been studied at different temperatures
and Mg2+ concentrations. Increasing the Mg2+ concentration in the denatured system significantly enhanced the inactivation and unfolding of the enzyme during thermal
inactivation. The analysis of the kinetic course of substrate reaction during thermal inactivation showed that at 47°C the
increased free Mg2+ concentration caused the inactivation rate to increase. Increasing the temperature strengthened the effect of Mg2+ on the thermal inactivation. Control experiment showed that this is not due to salt effect. The time course of fluorescence
emission spectra showed that the emission maximum for Mg2+-containing system was always higher than that of Mg2+-free system, and the higher temperature enhanced this difference. In addition, Mg2+also enhanced the unfolding rate of the enzyme at 47°C. The potential biological significance of these results are discussed. 相似文献
11.
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
12.
J E Norne H Szajn H Csopak P Reimarsson B Lindman 《Archives of biochemistry and biophysics》1979,196(2):552-556
The relation between Zn2+ binding of E. coli alkaline phosphatase and enzymatic activity and anion binding (using 35Cl NMR) has been investigated. The results suggest the existence of two forms of the enzyme with different zinc binding properties. The anion binding associated with the enzyme's function appears to be an amino acid residue and not the Zn2+ ions; furthermore, there is a rapid internal motion at the anion binding site. 35Cl relaxation studies in the presence of Mg2+ ions point to a marked interdependence of Mg2+ and Zn2+ binding. 相似文献
13.
Yurchenko JV Budilov AV Deyev SM Khromov IS Sobolev AY 《Molecular genetics and genomics : MGG》2003,270(1):87-93
A gene that codes for an alkaline phosphatase was cloned from the thermophilic bacterium Meiothermus ruber, and its nucleotide sequence was determined. The deduced amino acid sequence indicates that the enzyme precursor including the putative signal sequence is composed of 503 amino acid residues and has an estimated molecular mass of 54,229 Da. Comparison of the peptide sequence with that of the prototype alkaline phosphatase from Escherichia coli revealed conservation of the regions in the vicinity of the corresponding phosphorylation site and metal binding sites. The protein was expressed in E. coli and its enzymatic properties were characterized. In the absence of exogenously added metal ions, activity was negligible; to obtain maximal activity, addition of free Mg2+ ions was required. Zn2+ ions had an inhibitory effect on the activity of the M. ruber enzyme. The pH and temperature optima for activity were found to be 11.0 and 62°C, respectively. The enzyme was moderately thermostable: it retained about 50% activity after incubation for 6 h at 60°C, whereas at 80°C it was completely inactivated within 2 h. The Michaelis constant for cleavage of 4-nitrophenylphosphate was 0.055 mM. While having much in common with other alkaline phosphatases, the M. ruber enzyme presents some unique features, such as a very narrow pH range for activity and an absolute requirement for magnesium for activity.Communicated by G. P. Georgiev 相似文献
14.
Zheng Li Kimberly A. Stieglitz Anthony L. Shrout Yang Wei Robert M. Weis Boguslaw Stec Mary F. Roberts 《Protein science : a publication of the Protein Society》2010,19(2):309-318
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 μM for one ion with a KD of 38 μM 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. 相似文献
15.
Human placental alkaline phosphatase is a membrane-anchored protein. Entrapping the enzyme into a reverse micellar vesicle mimics the in vivo conditions and allows examination of the properties of the enzyme. Placental alkaline phosphatase is enzymatically active in Aerosol-OT/isooctane reverse micelles. Substantially different kinetic behavior of the enzyme has been observed in aqueous or reverse micellar systems. In aqueous solution, Mg2+ is a nonessential activator of the enzyme. In the experiments described in the present report Mg2+ was found to be an inhibitor for the enzyme in reverse micelles. This inhibition is presumably due to a time-dependent conformational change of the enzyme molecule, which resulted in a curvature in the recorder tracings of the enzyme assays. The Mg2+-induced conformational change of the enzyme was completely prevented by phosphate and partially reserved by EDTA. High concentrations of Zn2+ also strongly inhibited enzyme activity in both aqueous and reverse micellar solvent systems, presumably by occupying the Mg2+ (M3) site of the enzyme. However, binding of Zn2+ at the M3 site did not cause conformational change of the enzyme and the enzyme assay tracing was linear. The M3 site of the enzyme is proposed to have a modulatory role in vivo using magnesium ion as the modulator. 相似文献
16.
Protein phosphatase 2C (PP2C) family is characterized by requirement of metal cation for phosphatase activity. We previously
established that PPM1H is a cancer-associated member of the PP2C family. Here we further characterized the phosphatase activity
of PPM1H, focusing on its dependence on metal cation. PPM1H possesses the potential to dephosphorylate p-nitrophenyl phosphate (pNPP), casein and phosphopeptides. Interestingly, PPM1H shows the metal preference that is varied
depending on the substrate (substrate-dependent metal preference); PPM1H prefers Mn2+ when pNPP or phosphopeptides is used as a substrate. Meanwhile, a preference for Mg2+ is displayed by PPM1H with casein as a substrate. When both cations are added to the reaction, the degree of the effect is
always closer to that with Mn2+ alone, irrespective of the substrate. This preponderance of Mn2+ is explained by its greater affinity for PPM1H than Mg2+. From the literature the substrate-dependent metal preference appears to be shared by other PP2Cs. According to the crystal
structure, a binuclear metal center of PP2C plays an important role for coordinating the substrate and nucleophilic waters
in the active site. Therefore, the differences in the size, preferred geometry and coordination requirements between two metals,
in relation to the substrate, may be responsible for this intriguing property. 相似文献
17.
Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis 总被引:2,自引:0,他引:2
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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 (nH) 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. 相似文献
18.
A soluble protein phosphatase from the promastigote form of the parasitic protozoanLeishmania donovani was partially purified using Sephadex G-100, DEAE-cellulose and again Sephadex G-100 columns. The partially purified enzyme showed a native molecular weight of about 42, 000 in both Sephadex G-100 and sucrose density gradient centrifugation. The sedimentation constant, stokes radius and frictional ratio were found to be 3.43S, 2.8 nm and 1.20 respectively. The enzyme preferentially utilized phosphohistone as the best exogenous substrate. Mg2+ ions were essential for enzyme activity; among other metal ions Mn2+ can replace Mg2+ to a certain extent whereas Ca2+, Co2+ and Zn2+ could not substitute for Mg2+. The pH optimum of the enzyme was 6.5–7.5 and the temperature optimum 37°C. The apparent Km for phosphohistone was 7.14 M. ATP, ADP, inorganic phosphate and pyrophosphate had inhibitory effect on the enzyme activity whereas no inhibition was observed with sodium tartrate and okadaic acid. These results suggest thatL. donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C.Abbreviations PMSF
phenylmethylsulfonyl fluoride
- DTT
dithiothreitol
- TCA
trichloroacetic acid
- BSA
bovine serum albumin
- EDTA
ethylenediamine tetraacetic acid
- ATP
adenosine triphosphate
- ADP
adenosine diphosphate
- AMP
adenosine monophosphate
- EGTA
Ethyleneglycol-bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid 相似文献
19.
R.KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of Ca2+ and Mg2+
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KpnI REase recognizes palindromic sequence, GGTAC↓C, and forms complex in the absence of divalent metal ions, but requires the ions for DNA cleavage. Unlike most other REases, R.KpnI shows promiscuous DNA cleavage in the presence of Mg2+. Surprisingly, Ca2+ suppresses the Mg2+-mediated promiscuous activity and induces high fidelity cleavage. To further analyze these unique features of the enzyme, we have carried out DNA binding and kinetic analysis. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, Ca2+-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both Mg2+- and Mn2+-mediated reactions and was about three times slower with Ca2+. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg2+-mediated reactions unlike any other Type II REases, accounting for the promiscuous behavior. R.KpnI, thus displays properties akin to that of typical Type II REases and also endonucleases with degenerate specificity in its DNA recognition and cleavage properties. 相似文献
20.
Two different folding aids including α-cyclodextrin and Mg2+ ions were applied to alkaline phosphatase refolding. The refolding yield depending on concentration of each of the refolding
agents reached to 55 and 52% in the presence of 100 mM α-cyclodextrin and/or 5 mM Mg2+ ions, respectively. However, the refolding yield, mediated by combination of α-cyclodextrin and Mg2+ ions, was more than 96%. Replacement of Mg2+ ion with other type of ions which interact with α-cyclodextrin interfere with the function of cyclodextrin resulting in low
refolding yields. 相似文献