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1.
The kinetic affinity for CO2 of phosphoenolpyruvate PEP5 carboxykinase from Anaerobiospirillum succiniciproducens, an obligate anaerobe which PEP carboxykinase catalyzes the carboxylation of PEP in one of the final steps of succinate production from glucose, is compared with that of the PEP carboxykinase from Saccharomyces cerevisiae, which catalyzes the decarboxylation of oxaloacetate in one of the first steps in the biosynthesis of glucose. For the A. succiniciproducens enzyme, at physiological concentrations of Mn2+ and Mg2+, the affinity for CO2 increases as the ATP/ADP ratio is increased in the assay medium, while the opposite effect is seen for the S. cerevisiae enzyme. The results show that a high ATP/ADP ratio favors CO2 fixation by the PEP carboxykinase from A. succiniciproducens but not for the S. cerevisiae enzyme. These findings are in agreement with the proposed physiological roles of S. cerevisiae and A. succiniciproducens PEP carboxykinases, and expand recent observations performed with the enzyme isolated from Panicum maximum (Chen et al. (2002) Plant Physiology 128: 160–164).  相似文献   

2.
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   

3.
Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO2. They are activated by Mn2+, a metal ion that coordinates to the protein through the ?-amino group of a lysine residue, the N?-2-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the ?-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the ?-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn2+ in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn2+ affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn2+. In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.  相似文献   

4.
5.
Phosphoenolpyruvate (PEP) carboxykinase was purified 42-fold with a 25% yield from cell extracts of Ruminococcus flavefaciens by ammonium sulfate precipitation, preparative isoelectric focusing, and removal of carrier ampholytes by chromatography. The enzyme had a subunit molecular mass of ∼66.3 kDa (determined by mass spectrometry), but was retained by a filter having a 100-kDa nominal molecular mass cutoff. Optimal activity required activation of the enzyme by Mn2+ and stabilization of the nucleotide substrate by Mg2+. GDP was a more effective phosphoryl acceptor than ADP, while IDP was not utilized. Under optimal conditions the measured activity in the direction of PEP carboxylation was 17.2 μmol min–1 (mg enzyme)–1. The apparent K m values for PEP (0.3 mM) and GDP (2.0 mM) were 9- and 14-fold lower than the apparent K m values for the substrates of the back reaction (oxaloacetate and GTP, respectively). The data are consistent with the involvement of PEP carboxykinase as the primary carboxylation enzyme in the fermentation of cellulose to succinate by this bacterium. Received: 20 August 1996 / Accepted: 28 December 1996  相似文献   

6.
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   

7.
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   

8.
The effect of agitation and aeration on the growth and antibiotic production by Xenorhabdus nematophila YL001 grown in batch cultures were investigated. Efficiency of aeration and agitation was evaluated through the oxygen mass transfer coefficient (K L a). With increase in K L a, the biomass and antibiotic activity increased. Activity units of antibiotic and dry cell weight were increased to 232 U ml−1 and 19.58 g l−1, respectively, productivity in cell and antibiotic was up more than 30% when K L a increased from 115.9 h−1 to 185.7 h−1. During the exponential growth phase, DO concentration was zero, the oxygen supply was not sufficient. So, based on process analysis, a three-stage oxygen supply control strategy was used to improved the DO concentration above 30% by controlling the agitation speed and aeration rate. The dry cell weight and activity units of antibiotic were further increased to 24.22 g l−1 and 249 U ml−1, and were improved by 24.0% and 7.0%, compared with fermentation at a constant agitation speed and a constant aeration rate (300 rev min−1, 2.5 l min−1).  相似文献   

9.
Two extracellular tannin acyl hydrolases (TAH I and TAH II) produced by an Antarctic filamentous fungus Verticillium sp. P9 were purified to homogeneity (7.9- and 10.5-fold with a yield of 1.6 and 0.9%, respectively) and characterized. TAH I and TAH II are multimeric (each consisting of approximately 40 and 46 kDa sub-units) glycoproteins containing 11 and 26% carbohydrates, respectively, and their molecular mass is approximately 155 kDa. TAH I and TAH II are optimally active at pH of 5.5 and 25 and 20°C, respectively. Both the enzymes were activated by Mg2+and Br ions and 0.5–2.0 M urea and inhibited by other metal ions (Zn2+, Cu2+, K+, Cd2+, Ag+, Fe3+, Mn2+, Co2+, Hg2+, Pb2+ and Sn2+), anions, Tween 20, Tween 60, Tween 80, Triton X-100, sodium dodecyl sulphate, β-mercaptoethanol, α-glutathione and 4-chloromercuribenzoate. Both tannases more efficiently hydrolyzed tannic acid than methyl gallate. E a of these reactions and temperature dependence (at 0–30°C) of k cat, k cat/K m, ΔG*, ΔH* and ΔS* for both the enzymes and substrates were determined. The k cat and k cat/K m values (for both the substrates) were considerably higher for the combined preparation of TAH I and TAH II.  相似文献   

10.
Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni2+-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml−1 medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn2+, Ca2+, Fe2+, Mg2+ and Fe3+ ions stimulated the pectate lyase activity, but Cu2+ and Zn2+ inhibited it. The recombinant PelA had a V max of 77 μmol min−1 mg−1 and an apparent K m of 0.50 mg ml−1 for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.  相似文献   

11.
The bceA J gene from the cystic fibrosis isolate Burkholderia cenocepacia J2315 encodes a 56-kDa bifunctional protein, with phosphomannose isomerase (PMI) and guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP) activities, a new member of the poorly characterised type II PMI class of proteins. Due to the lack of homology between the type II PMIs and the human PMI, this class of proteins are being regarded as interesting potential targets to develop new antimicrobials. The BceAJ protein conserves the four typical motifs of type II PMIs: the pyrophosphorylase signature, the GMP active site, the PMI active site and the zinc-binding motif. After overproduction of BceAJ by Escherichia coli as a histidine tag derivative, the protein was purified to homogeneity by affinity chromatography. The GMP activity is dependent on the presence of Mg2+ or Ca2+ as cofactors, while the PMI activity uses a broader range of divalent ions, in the order of activation Mg2+ > Ca2+ > Mn2+ > Co2+ > Ni2+. The kinetic parameters K m, V max and K cat/K m for the PMI and GMP activities were determined. Results suggest that the enzyme favours the formation of GDP-mannose instead of mannose catabolism, thus channelling precursors to the formation of glycoconjugates.  相似文献   

12.
The DR2356 nudix hydrolase gene from Deinococcus radiodurans has been cloned and the product expressed as an 18 kDa histidine-tagged protein. The enzyme hydrolysed adenosine and diadenosine polyphosphates, always generating ATP as one of the initial products. ATP and other (deoxy)nucleoside triphosphates were also substrates, yielding (d)NDP and Pi as products. The DR2356 protein was most active at pH 8.6–9.0 and showed a strong preference for Mn2+ as activating cation. Mg2+ ions at 15 mM supported only 5% of the activity achieved with 2 mM Mn2+. K m and k cat values for diadenosine tetra-, penta- and hexaphosphates were 2.0, 2.4 and 1.1 μM and 11.4, 28.6 and 12.0 s−1, respectively, while for GTP they were 20.3 μM and 1.8 s−1, respectively. The K m for adenosine 5′-pentaphosphate was <1 μM. Expression analysis showed the DR2356 gene to be induced eight- to ninefold in stationary phase and in cells subjected to slow dehydration plus rehydration. Superoxide (but not peroxide) treatment and rapid dehydration caused a two-to threefold induction. The Mn-requirement and induction in stationary phase suggest that DR2356 may have a specific role in maintenance mode metabolism in stationary phase as Mn2+ accumulates.  相似文献   

13.
The chemical composition of rainwater is altered upon its passage through tree canopies. In order to investigate how rainwater chemistry is affected by canopy-dependent processes in characteristic forest types of Northwest German sandy lowland regions – oak–birch-forests, Betula pubescens Ehrh. swamp forests, and stands of Pinus sylvestris L. – comparative studies on the chemical composition of throughfall were carried out at seven forest sites, situated in close proximity within a nature reserve. Additionally, rainwater was sampled at three heathland sites for analysis of open-field precipitation and at three sites along an oak–birch-forest edge. Throughfall concentrations of most of the parameters analysed were significantly higher than open-field concentrations, especially with regard to electric conductivity, NH4-N, K+, and KMnO4-index. Ion concentrations in throughfall were the lowest in a 10-year-old stand of Betula pendula Roth. and Pinus sylvestris and in a Betula pubescens swamp forest and were highest beneath a stand of Pinus sylvestris. Except for Na+, Cl, and NO3, ion concentrations in both throughfall and open-field precipitation increased during the growing season (May–October). In throughfall, Ca2+, Mg2+, K+, and Mn2+ were strongly correlated. Enrichment ratios between throughfall and open-field deposition varied among sites and elements and were the highest for K‰+, Mg2‰+, and Mn2‰+. Estimates of canopy leaching indicated high leaching rates of K‰+ and Mn2‰+ and moderate leaching of Mg2‰+. The contribution of foliar leaching to throughfall deposition was higher at the deciduous than at the coniferous stands.  相似文献   

14.
An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The Km and Vmax values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg−1 protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu2+ and Hg2+ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.  相似文献   

15.
In a greenhouse study, with and without rice plants, of five flooded Philippine rice soils whose organic C (OC) content varied from 0.5 to 3.6%, incorporation ofSesbania rostrata, Azolla microphylla and rice straw affected the kinetics of soil solution NH 4 + −N, K+, Fe2+, Mn2+, Zn2+, and P. Sesbania and Azolla increased NH 4 + −N concentration above the control treatment, whereas rice straw depressed it. In all soils Azolla released less NH 4 + −N than Sesbania. The apparent net N release depended on the soil and ranged from 44–81% for Sesbania and 27–52% for Azolla. These effects persisted throughout the growth of IR36. Soil solution and exchangeable NH 4 + −N increased initially but levelled off between 30 to 80 days and between 20 to 40 days after flooding (DF), respectively. With rice, soil solution NH 4 + −N concentration, reached a peak at 15–40 DF and declined to very low levels (<4mg L−1). In the 3 soils of low OC content nitrogen derived from green manure ranged from 34–53% and the apparent revovery of added green manure N varied from 29–67%. Almost all N released from both Azolla and Sesbania were recovered in the rice plant in all soils except Concepcion with only 77%. The concentration of K+, Fe2+, Mn2+ and P in the soil solution were higher with rice straw than Sesbania and Azolla in all soils except Hanggan which showed no change in Fe2+ and Mn2+ but increased K+ and P. In general, rice straw, Sesbania and Azolla decreased Zn2+ concentration in all soils.  相似文献   

16.
The differences in pigment levels, photosynthetic activity and the chlorophyll fluorescence decrease ratio R Fd (as indicator of photosynthetic rates) of green sun and shade leaves of three broadleaf trees (Platanus acerifolia Willd., Populus alba L., Tilia cordata Mill.) were compared. Sun leaves were characterized by higher levels of total chlorophylls a + b and total carotenoids x + c as well as higher values for the weight ratio chlorophyll (Chl) a/b (sun leaves 3.23–3.45; shade leaves: 2.74–2.81), and lower values for the ratio chlorophylls to carotenoids (a + b)/(x + c) (with 4.44–4.70 in sun leaves and 5.04–5.72 in shade leaves). Sun leaves exhibited higher photosynthetic rates P N on a leaf area basis (mean of 9.1–10.1 μmol CO2 m−2 s−1) and Chl basis, which correlated well with the higher values of stomatal conductance G s (range 105–180 mmol m−2 s−1), as compared to shade leaves (G s range 25–77 mmol m−2 s−1; P N: 3.2–3.7 μmol CO2 m−2 s−1). The higher photosynthetic rates could also be detected via imaging the Chl fluorescence decrease ratio R Fd, which possessed higher values in sun leaves (2.8–3.0) as compared to shade leaves (1.4–1.8). In addition, via R Fd images it was shown that the photosynthetic activity of the leaves of all trees exhibits a large heterogeneity across the leaf area, and in general to a higher extent in sun leaves than in shade leaves.  相似文献   

17.
The acidifying effect of PtII on nucleobase –NH and –NH2 groups depends both on the site of metal coordination and on the efficiency of stabilization of the deprotonated nucleobase via intracomplex hydrogen bonding. Weakly acidic nucleobase protons with pK a values between 9 and 17 can be acidified by a single PtII to have pK a values which are well within the physiological pH range. This could open the possibility of an acid–base catalysis occurring at pH 7, with the metal–nucleobase entity functioning either as an acid or a base. Examples of PtII complexes studied here include, among others, mixed nucleobase systems of 1-methylcytosine and 1,9-dimethyladenine as well as a complex of the rare iminooxo tautomer of 1-methylcytosine having the metal bonded at N4.  相似文献   

18.
Summary The mechanisms underlying the pacemaker current in cardiac tissues is not agreed upon. The pacemaker potential in Purkinje fibers has been attributed to the decay of the potassium current I Kdd. An alternative proposal is that the hyperpolarization-activated current I f underlies the pacemaker potential in all cardiac pacemakers. The aim of this review is to retrace the experimental development related to the pacemaker mechanism in Purkinje fibers with reference to findings about the pacemaker mechanism in the SAN as warranted. Experimental data and their interpretation are critically reviewed. Major findings were attributed to K+ depletion in narrow extracellular spaces which would result in a time dependent decay of the inward rectifier current I K1. In turn, this decay would be responsible for a “fake” reversal of the pacemaker current. In order to avoid such a postulated depletion, Ba2+ was used to block the decay of I K1. In the presence of Ba2+ the time-dependent current no longer reversed and instead increased with time and more so at potentials as negative as −120 mV. In this regard, the distinct possibility needs to be considered that Ba2+ had blocked I Kdd (and not only I K1). That indeed this was the case was demonstrated by studying single Purkinje cells in the absence and in the presence of Ba2+. In the absence of Ba2+, I Kdd was present in the pacemaker potential range and reversed at E K. In the presence of Ba2+, I Kdd was blocked and I f appeared at potentials negative to the pacemaker range. The pacemaker potential behaves in a manner consistent with the underlying I Kdd but not with I f. The fact that I f is activated on hyperpolarization at potential negative to the pacemaker range makes it suitable as a safety factor to prevent the inhibitory action of more negative potentials on pacemaker discharge. It is concluded that the large body of evidence reviewed proves the pacemaker role of I Kdd (but not of I f) in Purkinje fibers.  相似文献   

19.
The Mg2+ ion-assisted activation mechanism of the active site Tyr8 of a human hematopoietic prostaglandin D2 synthase (H-PGDS) was studied by ultraviolet resonance Raman (UVRR) spectroscopy. Addition of Mg2+ to the native H-PGDS at pH 8.0 resulted in the Y8a Raman band of Tyr8 shifting from 1615 cm−1 to 1600 cm−1. This large shift to lower energy of the tyrosine Y8a vibrational mode is caused by the deprotonation of the tyrosine phenol group promoted by binding of Mg2+. Upon subsequent addition of glutathione (GSH), the Mg2+/H-PGDS solution showed the Tyr8 Raman band shifted to 1611 cm−1, which is 11 cm−1 higher than the frequency of the Mg2+ complex of H-PGDS, but 4 cm−1 lower than the Mg2+ free enzyme. These UVRR observations suggest that the deprotonated Tyr8 in the presence of Mg2+ is re-protonated by the abstraction of H+ from the thiol group of GSH, and that the re-protonated Tyr8 species forms a hydrogen bond with the thiolate anion of GSH. Density functional theory calculations on several model complexes of p-cresol were also performed, which suggested that the pKa and vibrational frequencies of the Tyr8 phenol group are affected by the degree and structure of hydration of the Tyr8 residue.  相似文献   

20.
We have developed NMR spectroscopic methods to investigate the tyrosines within Bacillus circulans xylanase (BcX). Four slowly exchanging buried tyrosine hydroxyl protons with chemical shifts between 7.5 and 12.5 ppm were found using a long-range 13C-HSQC experiment that exploits the 3JCH coupling between the ring 1Hη and 13Cε nuclei. The NMR signals from these protons were assigned via 13C-tyrosine selective labelling and a suite of scalar and 13C,15N-filtered/edited NOE correlation spectra. Of the fifteen tyrosines in BcX, only the buried Tyr79 and Tyr105 showed four distinct, rather than two averaged, signals from ring 13C–1H pairs, indicative of slow flipping on the chemical shift timescale. Ring flipping rate constants of ~10 and ~0.2 s−1 were measured for the two residues, respectively, using a 13C longitudinal exchange experiment. The hydrogen bonding properties of the Tyr79 and Tyr105 hydroxyls were also defined by complementary NOE and J-coupling measurements. The 1Hη hydrogen–deuterium exchange rate constants of the buried tyrosines were determined from 13C/15N-filtered spectra recorded as a function of pH. These exchange rate constants correspond to estimated protection factors of ~104–108 relative to a random coil tyrosine. The phenolic sidechain pK a values were also measured by monitoring their pH-dependent 13Cζ chemical shifts via 1Hε/δ(13Cε)13Cζ correlation spectra. Exposed tyrosines had unperturbed pK a values of ~10.2, whereas buried residues remained predominantly neutral at or even above pH 11. Combined with selective isotope labelling, these NMR experiments should prove useful for investigating the structural and electrostatic properties of tyrosines in many interesting proteins.  相似文献   

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