共查询到20条相似文献,搜索用时 15 毫秒
1.
We report the first kinetic characterization of human liver cytosolic GTP-dependent phosphoenolpyruvate carboxykinase (GTP-PEPCK), which plays a major role in the development of type 2 diabetes in human. In this work two recombinant forms of the enzyme were studied. One form had a His10-tag and the other was His-tag-free, and with one exception, both exhibited similar kinetic properties. When Mn2+ was used as the sole divalent cation, the His10-tagged enzyme, but not the His-tag-free enzyme, was increasingly inhibited at Mn2+ concentrations greater than 0.7 mM. This inhibition did not pose any problem in kinetic analysis, for within the relevant Mn2+ concentration range the His-tagged human PEPCK behaved almost identically to the tag-free enzyme. This property will bring simplicity and speed to purifying and studying multiple structural variants of this important enzyme. Apparent Km values of tag-free enzyme for phosphoenolpyruvate, GDP and bicarbonate were 450, 79 and 20,600 microM, respectively, while those for oxaloacetate and GTP were 4 and 23 microM, respectively, emphasizing the enzyme's gluconeogenic character. Bicarbonate (>100 mM) inhibited OAA-forming activity, which was a new observation with a GTP-PEPCK. The apparent Km for Mn2+ in the PEP-forming direction was 30-fold lower than that for the OAA-forming direction. Mn2+ and bicarbonate or CO2 might regulate the enzyme in vivo. 相似文献
2.
Miriam Escós Pedro Latorre Jorge Hidalgo Ramón Hurtado-Guerrero José Alberto Carrodeguas Pascual López-Buesa 《Biochemistry and Biophysics Reports》2016
The cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) plays a regulatory role in gluconeogenesis and glyceroneogenesis. The role of the mitochondrial isoform (PCK2) remains unclear. We report the partial purification and kinetic and functional characterization of human PCK2. Kinetic properties of the enzyme are very similar to those of the cytosolic enzyme. PCK2 has an absolute requirement for Mn2+ ions for activity; Mg2+ ions reduce the Km for Mn2+ by about 60 fold. Its specificity constant is 100 fold larger for oxaloacetate than for phosphoenolpyruvate suggesting that oxaloacetate phosphorylation is the favored reaction in vivo. The enzyme possesses weak pyruvate kinase-like activity (kcat=2.7 s?1). When overexpressed in HEK293T cells it enhances strongly glucose and lipid production showing that it can play, as the cytosolic isoenzyme, an active role in glyceroneogenesis and gluconeogenesis. 相似文献
3.
Chunxia Zhao Xin Zhan Sidney W. Whiteheart Michael G. Fried 《Analytical biochemistry》2010,399(2):237-245
The hexahistidine (His6)/nickel(II)-nitrilotriacetic acid (Ni2+-NTA) system is widely used for affinity purification of recombinant proteins. The NTA group has many other applications, including the attachment of chromophores, fluorophores, or nanogold to His6 proteins. Here we explore several applications of the NTA derivative, (Ni2+-NTA)2-Cy3. This molecule binds our two model His6 proteins, N-ethylmaleimide sensitive factor (NSF) and O6-alklyguanine-DNA alkyltransferase (AGT), with moderate affinity (K ∼ 1.5 × 106 M−1) and no effect on their activity. Its high specificity makes (Ni2+-NTA)2-Cy3 ideal for detecting His6 proteins in complex mixtures of other proteins, allowing (Ni2+-NTA)2-Cy3 to be used as a probe in crude cell extracts and as a His6-specific gel stain. (Ni2+-NTA)2-Cy3 binding is reversible in 10 mM ethylenediaminetetraacetic acid (EDTA) or 500 mM imidazole, but in their absence it exchanges slowly (kexchange ∼ 5 × 10−6 s−1 with 0.2 μM labeled protein in the presence of 1 μM His6 peptide). Labeling with (Ni2+-NTA)2-Cy3 allows characterization of hydrodynamic properties by fluorescence anisotropy or analytical ultracentrifugation under conditions that prevent direct detection of protein (e.g., high ADP absorbance). In addition, fluorescence resonance energy transfer (FRET) between (Ni2+-NTA)2-Cy3-labeled proteins and suitable donors/acceptors provides a convenient assay for binding interactions and for measurements of donor-acceptor distances. 相似文献
4.
Behm C. A. and Bryant C. 1982. Phosphoenolpyruvate carboxykinase from Fasciola hepatica. International Journal for Parasitology12: 271–278. The kinetic properties of a partially purified preparation of phosphoenolpyruvate carboxykinase (PEPCK) from F. hepatica were examined. The pH optimum for the carboxylation reaction is 5.8–6.2. The enzyme is more active with Mn2+ than Mg2+ and the Mn2+ saturation curve was sigmoid. Apparent Km values for the substrates GDP, IDP, PEP and HCO3? were determined and found to be in the same range as those reported for other helminths except that the enzyme is less sensitive to low PEP concentrations. GTP and ATP at 0.5 and 1.0 mM inhibit the enzyme; the GTP inhibition was greater in the presence of Mg2+ than Mn2+ and was competitive with GDP. It was concluded that the activity of PEPCK from F. hepatica is controlled by the concentration of reactants and the ambient pH, that the accumulation of GTP is a sensitive mechanism for inhibiting the carboxylation reaction and that PEPCK activity in the cytosol is likely to be favoured over that of pyruvate kinase except when pH is high and PEP concentration low. 相似文献
5.
Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)-1·min-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. Km values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (Ka) for Mg2+ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW
molecular weight
- PEP (Case)
phosphoenolpyruvate (carboxylase)
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis 相似文献
6.
Jabalquinto AM Laivenieks M González-Nilo FD Yévenes A Encinas MV Zeikus JG Cardemil E 《Journal of Protein Chemistry》2002,21(6):393-400
Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate, and carbon dioxide, and uses Mn2+ as the activating metal ion. The enzyme is a monomer and presents 68% identity with Escherichia coli PEP carboxykinase. Comparison with the crystalline structure of homologous E. coli PEP carboxykinase [Tari, L. W., Matte, A., Goldie, H., and Delbaere, L. T. J. (1997). Nature Struct. Biol.
4, 990–994] suggests that His225, Asp262, Asp263, and Thr249 are located in the active site of the protein, interacting with manganese ions. In this work, these residues were individually changed to Gln (His225) or Asn. The mutated enzymes present 3–6 orders of magnitude lower values of V
max/K
m, indicating high catalytic relevance for these residues. The His225Gln mutant showed increased K
m values for Mn2+ and PEP as compared with wild-type enzyme, suggesting a role of His225 in Mn2+ and PEP binding. From 1.5–1.6 Kcal/mol lower affinity for the 3(2)-O-(N-methylantraniloyl) derivative of adenosine diphosphate was observed for the His225Gln and Asp263Asn mutant A. succiniciproducens PEP carboxykinases, implying a role of His225 and Asp263 in nucleotide binding. 相似文献
7.
8.
9.
Jabalquinto AM Laivenieks M Cabezas M Zeikus JG Cardemil E 《Journal of Protein Chemistry》2002,21(7):443-445
Anaerobiospirillum succiniciproducens His225Gln, Asp262Asn, Asp263Asn, and Thr249Asn phosphoenolpyruvate carboxykinases were analyzed for their oxaloacetate decarboxylase, and pyruvate kinase–like activities. The His225Gln and Asp263Asn enzymes showed increased K
m values for Mn2+ and PEP compared with the native enzyme, suggesting a role of His225 and Asp263 in Mn2+ and PEP binding. No mayor alterations in K
m values for oxaloacetate were detected for the varied enzymes. Alterations of His225, Asp262, Asp263, or Thr249, however, did not affect the V
max of the secondary activities as much as they affected the V
max for the main reaction. The results presented in this communication suggest different rate-limiting steps for the primary reaction and the secondary activities. 相似文献
10.
Hsiang-Ling Chen Chen-Tien Chang Long-Liu Lin Tein-Yu Li Huei-Fen Lo 《World journal of microbiology & biotechnology》2009,25(2):323-330
To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His6-tagged EcDCP (His6-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity
by Ni2+-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His6-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His6-EcDCP was stimulated by 1.5 fold. The K
M and k
cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s−1, respectively. His6-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected
by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His6-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17%
α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme. 相似文献
11.
Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from
PEP, adenosine diphosphate and carbon dioxide, and uses Mn2+ as the activating metal ion. Comparison with the crystalline structure of homologous Escherichia coli PEP carboxykinase [Tari et al. (1997) Nature Struct. Biol.
4, 990–994] shows that Lys213 is one of the ligands to Mn2+ at the enzyme active site. Coordination of Mn2+ to a lysyl residue is not common and suggests a low pK
a value for the ε-NH2 group of Lys213. In this work, we evaluate the role of neighboring Phe216 in contributing to provide a low polarity microenvironment suitable to keep the ε-NH2 of Lys213 in the unprotonated form. Mutation Phe216Tyr shows that the introduction of a hydroxyl group in the lateral chain of the
residue produces a substantial loss in the enzyme affinity for Mn2+, suggesting an increase of the pK
a of Lys213. In agreement with this interpretation, theoretical calculations indicate an alkaline shift of 2.8 pH units in the pK
a of the ε-amino group of Lys213 upon Phe216Tyr mutation. 相似文献
12.
R K Prichard 《International journal for parasitology》1976,6(3):227-233
F. hepatica pyruvate kinase and phosphoenolpyruvate (PEP) carboxykinase were found to have properties of regulatory enzymes in the dissimilation of PEP and the control of metabolic flow. Mn2+ and K+ were required for pyruvate kinase activity. In the presence of fructose-1, 6-diphosphate (FDP), Mg2+ could substitute for Mn2+. FDP caused a 4-fold increase in the Mn2+ activated pyruvate kinase activity. This was accompanied by a 12-fold decrease in apparent Km(PEP) and a 3-fold decrease in apparent Km (ADP). ATP markedly inhibited F. hepatica pyruvate kinase, but this inhibition was relieved by FDP. Estimates of metabolic levels indicated that the pyruvate kinase is saturated with PEP and ADP in vivo, but will be highly sensitive to fluctuations in the physiological concentrations of FDP and ATP. NADH doubled the activity of the PEP carboxykinase reaction and decreased the apparent Km (PEP) for this enzyme 3-fold. While the maximal activity of the PEP carboxykinase reaction was substantially higher than the pyruvate kinase reaction, the steady state concentration of PEP suggests that the PEP carboxykinase will not be saturated with this substrate. 相似文献
13.
In Acetobacter aceti growing on pyruvate as the only source of carbon and energy, oxaloacetate (OAA) is produced by a phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31). The enzyme was purified 122-fold and a molecular weight of about 380,000 was estimated by gel filtration.The optimum pH was 7.5 and the K
m
values for PEP and NaHCO3 were 0.49 mM and about 3 mM, respectively. The enzyme needed a divalent cation; the K
m
for Mn2+, Co2+ and Mg2+ were 0.12, 0.26 and 0.77 mM, respectively. Maximal activity was only obtained with Mg2+. Mn2+ and Co2+ became inhibitory at high concentrations.The activity was inhibited by succinate and, to a lesser extent, by fumarate, citrate, -ketoglutarate, aspartate and glutamate.As compared with the corresponding enzyme from A. xylinum, the PEP carboxylase of A. aceti showed the following differences: a) It had an absolute requirement for acetyl CoA (K
a
0.18 mM) or propionyl CoA (K
a
0.2 mM). b) It was not affected by ADP. c) It was sensitive to thiol blocking agents.Abbreviations PEP
phosphoenolpyruvate
- OAA
oxaloacetate
- MW
molecular weight
- TEMG buffer
50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl2, 1 mM glutathione
- HEPES
N-2-hydroxyethylpiperazine-N-ethanesulfonic acid 相似文献
14.
Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Mycobacterium smegmatis has been purified to homogeneity through a seven-step procedure with a yield of 16% and specific activity of 220 units/mg protein. The purified enzyme had a molecular weight of 230,700 and was composed of four subunits with identical molecular weights of 57,540. Analysis of amino acid composition revealed a low content of aromatic amino acids. The enzyme exhibited sigmoidal kinetics of varying concentrations of phosphoenolpyruvate, the degree of cooperativity and S0.5v value for phosphoenolpyruvate being strongly dependent on the pH of the reaction mixture. Among the nucleoside diphosphates acting as substrate for pyruvate kinase, ADP was the best phosphate acceptor, as judged by its lowest Km value. The enzyme showed an absolute requirement for divalent cations (either Mg2+ or Mn2+), but monovalent cations were not necessary for activity. Other divalent cations inhibited the Mg2+-activated enzyme to varying degrees (Ni2+ > Zn2+ > Cu2+ > Ca2+ > Ba2+). The differences in the kinetic responses of the enzyme to Mg2+ and Mn2+ are discussed. 相似文献
15.
Tao Zhang Yujie Guo Hao Zhang Wanmeng Mu Ming Miao Bo Jiang 《Process Biochemistry》2013,48(4):663-668
An l-ornithine high producing strain Bacillus thuringiensis SK20.001 was screened by our laboratory. An intracellular arginase used to biosynthesize l-ornithine from the strain was purified and characterized. The final specific arginase activity was 589.2 units/mg, with 70.1 fold enrichment and 22.4% recovery. The molecular weight of the enzyme was approximately 33,000 Da as evaluated by SDS-PAGE and 191,000 Da as determined by gel filtration. The enzyme had an optimum pH of 10.0 and an optimum temperature of 40 °C. It was stable from pH 8.0–12.0 and <50 °C without Mn2+. The presence of Mn2+ and Ni2+ had strong effects on the enzyme activity, and Mn2+ significantly increased the thermal stability of the enzyme. The arginase was slightly inhibited by Ca2+, Fe2+ and Zn2+. Trp, Asp, Glu, Tyr, and Arg residues were directly involved in the arginase activity evaluated by chemical modifications. The Km and Vmax for l-arginine were estimated to be 15.6 mM and 538.9 μmol/min/mg. The biosynthesis yield of l-ornithine was 72.7 g/L with the enzyme. 相似文献
16.
Trehalose is a unique disaccharide capable of protecting proteins against environmental stress. A novel trehalose synthase (TreS) gene from Rhodococcus opacus was cloned and expressed in Escherichia coli Top10 and BL21 (DE3) pLysS, respectively. The recombinant TreS showed a molecular mass of 79 kDa. Thin layer chromatography (TLC) result suggested that this enzyme had the ability to catalyze the mutual conversion of maltose and trehalose. Moreover, high-performance liquid chromatography (HPLC) result suggested that glucose appeared as a byproduct with a conversion rate of 12 %. The purified recombinant enzyme had an optimum temperature of 25 °C and pH optimum around 7.0. Kinetic analysis revealed that the K m for trehalose was around 98 mM, which was a little higher than that of maltose. The preferred substrate of TreS was maltose according to the analysis of k cat/K m. Both 1 and 10 mM of Hg2+, Cu2+ and Al3+ could inhibit the TreS activity, while only 1 mM of Ca2+ and Mn2+ could increase its activity. Five amino acid residues, Asp244, Glu286, Asp354, His147 and His353, were shown to be conserved in R. opacus TreS, which were also important for α-amylase family enzyme catalysis. 相似文献
17.
Phosphopentomutase (PPM) catalyzes the interconversion of α-d-(deoxy)-ribose 1-phosphate and α-d-(deoxy)-ribose 5-phosphate. We developed a coupled or uncoupled enzymatic assay with an enzyme nucleoside phosphorylase for determining PPM activities on d-ribose 5-phosphate at a broad temperature range from 30 to 90 °C. This assay not only is simple and highly sensitive but also does not require any costly special instrument. Via this technology, an open reading frame TM0167 from a thermophilic bacterium Thermotoga maritima putatively encoding PPM was cloned. The recombinant PPM was overexpressed in Escherichia coli Rosetta. This enzyme has the highest activity at 90 °C. MnCl2 (0.1 mM) and 50 μM α-d-glucose 1,6-bisphosphate are cofactors. The kinetic parameters of Km and kcat are 1.2 mM and 185 s−1 at 90 °C, respectively. The enzyme has a half-life time of up to 156 min at 90 °C. This enzyme is the most active and thermostable PPM reported to date. 相似文献
18.
《Process Biochemistry》2010,45(7):1088-1093
An extracellular thermostable α-galactosidase from Aspergillus parasiticus MTCC-2796 was purified 16.59-fold by precipitation with acetone, followed by sequential column chromatography with DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found to be a monomeric protein with a molecular weight of about 67.5 kDa. The purified enzyme showed optimum activity against o-nitrophenyl-α-d-galactopyranoside (oNPG) at pH 5.0 and a temperature of 50 °C. The enzyme was thermostable, showing complete activity even after heating at 65 °C for 30 min. The enzyme showed strict substrate specificity for α-galactosides and hydrolyzed oNPG (Km = 0.83 mM), melibiose (Km = 2.48 mM) and raffinose (Km = 5.83 mM). Among metal ions and reagents tested, Ca2+ and K+ enhanced the enzymatic activity, but Mg2+, Mn2+, ethylenediaminetetraacetic acid (EDTA) and 2-mercaptoethanol showed no effect, while Ag+, Hg2+ and Co2+ strongly inhibited the activity of the enzyme. The enzyme catalyzed the transglycosylation reaction for the synthesis of melibiose. 相似文献
19.
António Carloto María Jesús Costas José Carlos Cameselle Alexander G. McLennan João Meireles Ribeiro 《Biochimica et Biophysica Acta (BBA)/General Subjects》2006
Free ADP-ribose is a putative second messenger and also a potentially toxic compound due to its non-enzymic reactivity towards protein side chains. ADP-ribose hydrolysis is catalysed by NDP-sugar/alcohol pyrophosphatases of differing specificity, including a highly specific, low-Km ADP-ribose pyrophosphatase. In humans, a submicromolar-Km ADP-ribose pyrophosphatase has been purified from placenta, while recombinant NUDT9 has been described as a similarly specific enzyme with a nudix motif, but with a 102–103 higher Km. Here, a comparative study of both proteins is presented showing that they are in fact enzymically indistinguishable; crucially, they both have submicromolar Km for ADP-ribose. This study firmly supports the view that the ADP-ribose pyrophosphatase present in human tissues is a product of the NUDT9 gene. In addition, this study reveals previously unknown properties of both enzyme forms. They display the same, differential properties in the presence of Mg2+ or Mn2+ as activating cations with respect to substrate specificity, ADP-ribose saturation kinetics, and inhibition by fluoride. Treatment with H2O2 alters the Mg2+/Mn2+ responses and increases the Km values for ADP-ribose, changes that are reversed by DTT. The results are discussed in relation to the proposed roles for ADP-ribose in oxidative/nitrosative stress and for ADP-ribose pyrophosphatase as a protective enzyme whose function is to limit the intracellular accumulation of ADP-ribose. 相似文献
20.
Structure and Function of 2,3-Dimethylmalate Lyase, a PEP Mutase/Isocitrate Lyase Superfamily Member
Buvaneswari Narayanan Weiling Niu Henk-Jan Joosten Remko K.P. Kuipers Debra Dunaway-Mariano Osnat Herzberg 《Journal of molecular biology》2009,386(2):486-26696
The Aspergillus niger genome contains four genes that encode proteins exhibiting greater than 30% amino acid sequence identity to the confirmed oxaloacetate acetyl hydrolase (OAH), an enzyme that belongs to the phosphoenolpyruvate mutase/isocitrate lyase superfamily. Previous studies have shown that a mutant A. niger strain lacking the OAH gene does not produce oxalate. To identify the function of the protein sharing the highest amino acid sequence identity with the OAH (An07g08390, Swiss-Prot entry Q2L887, 57% identity), we produced the protein in Escherichia coli and purified it for structural and functional studies. A focused substrate screen was used to determine the catalytic function of An07g08390 as (2R,3S)-dimethylmalate lyase (DMML): kcat = 19.2 s− 1 and Km = 220 μM. DMML also possesses significant OAH activity (kcat = 0.5 s− 1 and Km = 220 μM). DNA array analysis showed that unlike the A. niger oah gene, the DMML encoding gene is subject to catabolite repression. DMML is a key enzyme in bacterial nicotinate catabolism, catalyzing the last of nine enzymatic steps. This pathway does not have a known fungal counterpart. BLAST analysis of the A. niger genome for the presence of a similar pathway revealed the presence of homologs to only some of the pathway enzymes. This and the finding that A. niger does not thrive on nicotinamide as a sole carbon source suggest that the fungal DMML functions in a presently unknown metabolic pathway. The crystal structure of A. niger DMML (in complex with Mg2+ and in complex with Mg2+ and a substrate analog: the gem-diol of 3,3-difluoro-oxaloacetate) was determined for the purpose of identifying structural determinants of substrate recognition and catalysis. Structure-guided site-directed mutants were prepared and evaluated to test the contributions made by key active-site residues. In this article, we report the results in the broader context of the lyase branch of the phosphoenolpyruvate mutase/isocitrate lyase superfamily to provide insight into the evolution of functional diversity. 相似文献