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1.
Antasar Ali D. Claire Wathes Angelina Swali Helena Burns Shamus Burns 《Biochemistry and Biophysics Reports》2017
BackgroundHexokinase and glucokinase enzymes are ubiquitously expressed and use ATP and ADP as substrates in mammalian systems and a variety of polyphosphate substrates and/or ATP in some eukaryotic and microbial systems. Polyphosphate synthesising or utilizing enzymes are widely expressed in microbial systems but have not been reported in mammalian systems, despite the presence of polyphosphate in mammalian cells. Only two micro-organisms have previously been shown to express an enzyme that uses polyphosphate exclusively.MethodsA variety of experimental approaches, including NMR and NAD-linked assay systems were used to conduct a biochemical investigation of polyphosphate dependent glucokinase activity in mammalian tissues.ResultsA novel mammalian glucokinase, highly responsive to hexametaphosphate (HMP) but not ATP or ADP as a phosphoryl donor is present in the nuclei of mammalian hepatocytes. The liver enzyme exhibited sigmoidal kinetics with respect to glucose with a S0.5 of 12 mM, similar to the known kinetics of mammalian ATP-glucokinase. The Km for HMP (0.5 mM) was also similar to that of phosphoryl donors for mammalian ATP-glucokinases. The new enzyme was inhibited by several nucleotide phosphates.ConclusionsWe report the discovery of a polyphosphate-dependent enzyme system in mammalian cells with kinetics similar to established ATP-dependent glucokinase, also known to have a nuclear location. The kinetics suggest possible regulatory or redox protective roles.General significanceThe role of polyphosphate in mammalian systems has remained an enigma for decades, and the present report describes progress on the significance of this compound in intracellular metabolism in mammals. 相似文献
2.
Kousaku Murata Jyoji Kato Ichiro Chibata 《Bioscience, biotechnology, and biochemistry》2013,77(12):2221-2226
The activities of polyphosphate glucokinase which synthesizes glucose-6-phosphate from glucose and metaphosphate were found in some microorganisms. This enzyme occurred most abundantly in Micrococcus and Achromobacter species and less in Brevibacterium, Aerobacter and Alcaligenes species. The distribution pattern of this enzyme was closely similar to that of polyphosphate NAD kinase. Polyphosphate glucokinase in A. butyri was different from ATP-dependent glucokinase in some enzymatic properties. The potent phosphoryl donor for this enzyme was metaphosphate, and other chain or ring phosphate polymers were not utilized by this enzyme. 相似文献
3.
Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate:glucose phosphotransferase) was covalently coupled to collagen-coated silica gel beads. The immobilized enzyme, as a packed-bed reactor, was used to determine glucose in serum and other samples. The method was based on a spectrophotometric measurement of NADPH produced by two consecutive reactions, similar to the hexokinase method. The described approach takes advantage of the greater stability of polyphosphate compared to that of ATP, the greater specificity of polyphosphate glucokinase versus that of hexokinase, and the reusability of the immobilized enzyme. Linearity, precision, and accuracy of the method were tested and found to be very good. The results were linear between 10 and 50 nmol of glucose in a 50-microliter sample and the coefficient of variation was less than 4% in five successive determinations. The recovery of glucose was about 100% after calibration of the method. The results of the measurements correlated well with those obtained with soluble polyphosphate glucokinase (r = 0.997, y = 1.036x - 0.016). The immobilized-enzyme reactor showed good operational stability during a month of use, losing about 12% of its initial activity. 相似文献
4.
Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate glucose phosphotransferase) has been partially purified (960-fold) from Propionibacterium shermanii. Throughout the purification, the ratio of polyphosphate glucokinase activity to ATP glucokinase activity remained approximately constant at 4 to 1. It is considered that both activities are catalyzed by the same protein. The mechanism of utilization of polyphosphate by polyphosphate glucokinase was investigated using polyphosphates of limited sizes that were isolated following gel electrophoresis of commercial heterogeneous polyphosphates. The results show that with long chain polyphosphates, the reaction proceeds by a processive type mechanism, and with short polyphosphates, it is nonprocessive. The Km for polyphosphate of chain length 724 is 2 X 10(-3) microM and increases with a decrease in chain length to 3.7 X 10(-2) microM at chain length 138. Subsequently, there is a very rapid increase of Km and at chain length 30 the Km is 4.3 microM. The rapid change in Km coincides with the shift in mechanism from the processive type mechanism in which there apparently is successive phosphorylation prior to release from the enzyme to a nonprocessive process in which the polyphosphate is released from the enzyme after each transfer. During the nonprocessive process, there is preferential utilization of the longer species. The Vmax is relatively constant with shorter polyphosphates but decreases with chain lengths longer than 347. In the cell, as a consequence of the low Km, the long chain polyphosphates probably are used preferentially to phosphorylate glucose. 相似文献
5.
In cell-free extracts of Acinetobacter strain 210A polyphosphate: AMP phosphotransferase and adenylate kinase activity was measured. Polyphosphate glucokinase and polyphosphate dependent NAD kinase were not detected. The specific activity of polyphosphate: AMP phosphotransferase was found to be 43 nmol · min-1 · mg-1 protein in presence of 1 mmol · l-1 AMP. The adenylate kinase reaction had an equilibrium constant ([ATP] [AMP] [ADP]-2) of 0.7, an activity of 54 nmol · min-1 · mg-1 protein, and was almost completely inhibited by 0.3 mM P1,P5-di(adenosine-5)-pentaphosphate. ATP was formed through the combined action of polyphosphate: AMP phosphotransferase and adenylate kinase in cell-free extracts from bacterial polyphosphate and from chemically prepared polyphosphate (Graham's salt). A spectrophotometric method for the continuous monitoring of polyphosphate: AMP phosphotransferase is also presented.Abbreviations Ap5A
P1,P5-di(adenosine-5)-pentaphosphate
- G6P-DH
D-glucose-6-phosphate dehydrogenase
- HK
hexokinase
- AEC
adenylate energy charge
- U
units (converting 1 mol · min-1) 相似文献
6.
Kerstin Bark Peter Kämpfer Alexander Sponner Wolfgang Dott 《FEMS microbiology letters》1993,107(2-3):133-138
Abstract Eleven isolates obtained from a laboratory sewage treatment plant, most of them presumptively assigned to the coryneform genera Curtobacterium and Aureobacterium were studied for the presence of intracellular polyphosphates and polyphosphate dependent enzymes. All isolates stored polyphosphates and showed adenylate kinase activities ranging from 64 to 815 mU mg−1 . Polyphosphate:AMP phosphotransferase could only be detected in one isolate. Three isolates showed a polyphosphate kinase activity also in minor amounts from 15 to 17 mU mg−1 . A polyphosphate dependent NAD or 3-phosphoglycerate kinase could not be detected. Polyphosphate glucokinase activity was measured in cell-free extracts of nine isolates ranging from 2 to 376 mU mg−1 . Three isolates showed in addition to the polyphosphate glucokinase, a glucose-6-phosphate-dependent NAD kinase. For the regeneration of NADP from NAD and polyphosphate, this enzyme system may give the isolates a distinct competitive advantage, especially for anabolic processes. The polyphosphate-dependent enzymes reported here may play an additional role in the complex process of 'biological' phosphate removal from wastewater. 相似文献
7.
The activity of ATP-glucokinase and of polyphosphate glucokinase was examined during growth of the actinomyceteStreptomyces aureofaciens 8425 under conditions of intense chlortetracycline (CTC) synthesis. ATP-glucokinase was active in the strain only during
the logarithmic phase of culture growth; the activity of polyphosphate glucokinase appears only at the end of the logarithmic
phase of growth and rises in parallel with the rate of CTC biosynthesis in the stationary phase. During the rise of activity
of polyphosphate glucokinase and of CTC biosynthesis the cells accumulate sugar phosphates, mainly glucose-6-phosphate. It
appears that the biosynthesis of CTC inStreptomyces aureofaciens takes place at the expense of glycolysis, using up the high-energy phosphate of high-molecular polyphosphates. 相似文献
8.
The hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324 has been shown to degrade starch via glucose using a modified Embden-Meyerhof pathway. The first enzyme of this pathway, ADP-dependent glucokinase, was purified 600-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of 50 kDa. It had a temperature optimum at 83 °C and showed a significant thermostability up to 100 °C. The enzyme was highly specific for ADP and glucose as substrates; it did not use ATP, CDP, UDP, or GDP as phosphoryl donors, or mannose, fructose and fructose 6-phosphate as phosphoryl acceptors (at 80 °C). Only glucosamine was phosphorylated at significant rates. The apparent Km values for ADP and glucose (at 50 °C) were 0.07 mM and 0.78 mM, respectively; the apparent Vmax value was about 50 U/mg at 50 °C and 350 U/mg at 80 °C. Divalent cations were required for maximal activity; Mn2+, Mg2+ and Ca2+, which were most effective, could be replaced partially by Cu2+, Ni2+, Co2+ and Zn2+. The N-terminal amino acid sequence (42 amino acids) of ADP-dependent glucokinase was almost identical to that of ADP-dependent glucokinase from Thermococcus litoralis. In the genome of the closely related Archaeoglobus fulgidus strain VC16 a homologous gene for ADP-dependent glucokinase could not be identified. 相似文献
9.
Steffen N. Lindner Sandra Knebel Srinivas R. Pallerla Siegfried M. Schoberth Volker F. Wendisch 《Applied microbiology and biotechnology》2010,87(2):703-713
The Corynebacterium glutamicum gene cg2091 is encoding a polyphosphate (PolyP)/ATP-dependent glucokinase (PPGK). Previous work demonstrated the association
of PPGK to PolyP granules. The deduced amino acid sequence of PPGK shows 45% sequence identity to PolyP/ATP glucomannokinase
of Arthrobacter sp. strain KM and 50% sequence identity to PolyP glucokinase of Mycobacterium tuberculosis H37Rv. PPGK from C. glutamicum was purified from recombinant Escherichia coli. PolyP was highly preferred over ATP and other NTPs as substrate and with respect to the tested PolyPs differing in chain
length; the protein was most active with PolyP75. Gel filtration analysis revealed that PolyP supported the formation of homodimers of PPGK and that PPGK was active as a
homodimer. A ppgK deletion mutant (ΔppgK) showed slowed growth in minimal medium with maltose as sole carbon source. Moreover, in minimal medium containing 2 to 4%
(w/v) glucose as carbon source, ΔppgK grew to lower final biomass concentrations than the wild type. Under phosphate starvation conditions, growth of ΔppgK was reduced, and growth of a ppgK overexpressing strain was increased as compared to wild type and empty vector control, respectively. Thus, under conditions
of glucose excess, the presence of PPGK entailed a growth advantage. 相似文献
10.
Paul N. Ulrich Noelia Lander Samarchith P. Kurup Laura Reiss Jessica Brewer Lia C. Soares Medeiros Kildare Miranda Roberto Docampo 《The Journal of eukaryotic microbiology》2014,61(2):155-165
Polyphosphate is a polymer of inorganic phosphate found in both prokaryotes and eukaryotes. Polyphosphate typically accumulates in acidic, calcium‐rich organelles known as acidocalcisomes, and recent research demonstrated that vacuolar transporter chaperone 4 catalyzes its synthesis in yeast. The human pathogens Trypanosoma brucei and T. cruzi possess vacuolar transporter chaperone 4 homologs. We demonstrate that T. cruzi vacuolar transporter chaperone 4 localizes to acidocalcisomes of epimastigotes by immunofluorescence and immuno‐electron microscopy and that the recombinant catalytic region of the T. cruzi enzyme is a polyphosphate kinase. RNA interference of the T. brucei enzyme in procyclic form parasites reduced short chain polyphosphate levels and resulted in accumulation of pyrophosphate. These results suggest that this trypanosome enzyme is an important component of a polyphosphate synthase complex that utilizes ATP to synthesize and translocate polyphosphate to acidocalcisomes in insect stages of these parasites. 相似文献
11.
Strictly polyphosphate-dependent glucokinase in a polyphosphate-accumulating bacterium,Microlunatus phosphovorus
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Tanaka S Lee SO Hamaoka K Kato J Takiguchi N Nakamura K Ohtake H Kuroda A 《Journal of bacteriology》2003,185(18):5654-5656
ATP-dependent glucokinase is suggested to have evolved from a hypothetical polyphosphate (polyP)-dependent glucokinase (polyP-GK) via a bifunctional polyP/ATP glucokinase (polyP/ATP-GK). Here we showed that polyP-GK is present in a polyP-accumulating bacterium, Microlunatus phosphovorus. The polyP-GK produced glucose-6-P(i) from glucose and polyP, but it could not phosphorylate glucose with ATP. The polyP-GK was most closely related to the polyP/ATP-GK of Mycobacterium tuberculosis. 相似文献
12.
《Bioscience, biotechnology, and biochemistry》2013,77(11):2322-2324
SCO5059, encoded in Streptomyces coelicolor A3(2), was identified as a polyphosphate glucokinase. The K m values of SCO5059 for glucose and polyphosphate (poly(P)6) were estimated to be 12 and 4 µM, respectively, and the k cat value was 0.3 s?1 at pH 7.7 at 28 °C. SCO5059 homologs are highly conserved among Streptomyces, and can work as polyphosphate glucokinase as well. 相似文献
13.
Cell extracts of Fibrobacter succinogenes subsp. succinogenes S85 phosphorylated glucose with a GTP-dependent glucokinase. The enzyme showed little activity with ATP (12% of that with GTP). Of other phosphate donors tested, only dGTP and ITP gave high glucokinase activities. Dialyzed extracts required Mg+2 and K+ for maximal activity. In potassium phosphate buffer, glucokinase showed maximum activity at pH 7.5 with glucose-6-phosphate dehydrogenase as the coupling enzyme. In this assay, glucokinase was active with glucose (100%), 2-deoxy-d-glucose (40%), and mannose (20%). Partially purified glucokinase had a molecular weight of 82,000 and a pl of 4.82. Double-reciprocal plots of substrate concentration versus velocity were linear and the enzyme had apparent Km values of 55 M for glucose and 72 M for GTP. Dialyzed cell extracts of Fibrobacter intestinalis C1A also contained a GTP-dependent glucokinase that showed little activity with ATP. Potassium also stimulated the activity of this enzyme. These results suggest that this unusual glucokinase may be characteristic of the genus Fibrobacter.Abbreviations
CHES
cyclohexylaminoethanesulfonic acid
-
GK
glucokinase
-
PEP
phosphoenolpyruvate
Published with the approval of the Director of the North Dakota Agricultural Experiment Station as journal article no. 2186 相似文献
14.
Douglas L. Coleman 《Biochemical genetics》1977,15(3-4):297-305
Inbred strains of mice were surveyed for liver glucokinase activity. Mice of all strains studied could be distributed into three groups with high, intermediate, and low levels of enzyme activity. Genetic analysis using crosses and backcrosses with prototype high (C3H/HeJ) and low (RF/J) strains revealed that glucokinase activity was controlled by a single gene. The name glucokinase and gene symbol Gk are suggested for this gene. The Gk
a allele designates the strain with high glucokinase activity, while Gk
b represents the allele in the strain with the low enzyme activity. The interaction of fasting and diabetes on the activity of glucokinase in these two strains is described.Supported in part by United States Public Health Service Research Grant CA 05873 from the National Cancer Institute. The Jackson Laboratory is fully accredited by the American Association for the Accreditation of Laboratory Animal Care. 相似文献
15.
Hehuan Liao Suwan Myung Y.-H. Percival Zhang 《Applied microbiology and biotechnology》2012,93(3):1109-1117
The discovery of stable and active polyphosphate glucokinase (PPGK, EC 2.7.1.63) would be vital to cascade enzyme biocatalysis
that does not require a costly ATP input. An open reading frame Tfu_1811 from Thermobifida fusca YX encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK)
was overexpressed in Escherichia coli. Mg2+ was an indispensible activator. This enzyme exhibited the highest activity in the presence of 4 mM Mg2+ at 55°C and pH 9.0. Under its suboptimal conditions (pH 7.5), the k
cat and K
m values of CBM-PPGK on glucose were 96.9 and 39.7 s−1 as well as 0.77 and 0.45 mM at 37°C and 50°C respectively. The thermoinactivation of CBM-PPGK was independent of its mass
concentration. Through one-step enzyme purification and immobilization on a high-capacity regenerated amorphous cellulose,
immobilized CBM-PPGK had an approximately eightfold half lifetime enhancement (i.e., t
1/2 = 120 min) as compared to free enzyme at 50°C. To our limited knowledge, this enzyme was the first thermostable PPGK reported.
Free PPGK and immobilized CBM-PPGK had total turnover number values of 126,000 and 961,000 mol product per mol enzyme, respectively,
suggesting their great potential in glucose-6-phosphate generation based on low-cost polyphosphate. 相似文献
16.
17.
O. Assemat M. Antoine J.-M. Fourquez M. Wierzbicki Y. Charton P. Hennig F. Perron-Sierra G. Ferry J.A. Boutin M.-A. Delsuc 《Analytical biochemistry》2015
Human hexokinase enzyme IV (EC 2.7.1.1) catalyzes the phosphorylation of glucose and regulates the level of glucose. This enzyme exhibits strong positive cooperativity due to an allosteric transition between an inactive form and a closed active form. This form can be stabilized by activators and, thus, can increase its turnover by a kinetic memory effect characterized by a slow decay to the inactive state. The structural details of this kinetic allostery are known. Several synthetic activators have been reported. We present a preliminary nuclear magnetic resonance (NMR) screening of a chemical library in search of molecules with some affinity for glucokinase (GK). The library, composed of eight molecules with known activity as well as molecules that display no interaction, has been tested using the FAXS (fluorine chemical shift anisotropy and exchange for screening) method, based on monitoring the R2 relaxation of the 19F spin. To ensure a valid interaction measurement, the enzyme was placed in the presence of glucose and magnesium. The binding signal of one known fluorinated ligand was measured by determining the displacement of the known ligand. This simple measure of the 19F signal intensity after an 80-ms spin echo correlates nicely with the EC50, opening a route for NMR screening of GK activators. 相似文献
18.
Trichodesmium tenue Wille (1904) was examined using transmission electron microscopy to determine the role of carbohydrate, phosphorus, and nitrogen storage in buoyancy regulation. Carbohydrate storage area (mean = 2.06 ± 0.61 [SE] μm2; 6.62% of total cell area) in negatively buoyant colonies (NBCs) was significantly higher (P < 0.001) than in positively buoyant colonies (PBCs) (mean = 0.38 ± 0.06 μm2; 0.73%). Distinct diel periodicity of carbohydrate content was found in NBCs demonstrated by an increase from darkness to afternoon. Polyphosphate content was significantly higher (P < 0.001) in NBCs, with a mean of 0.44± 0.10 μm2 (1.54%), as compared to PBCs, with a mean of 0.14 ± 0.05 μm2 (0.24%). Polyphosphate content increased in NBCs from morning to evening, and PBCs had a 10% decrease from morning to afternoon. Calculations indicated that averaged effects of polyphosphate on increased cell density is approximately 20% of that from carbohydrate accumulation. Density contribution due to ballast weight of carbohydrate and polyphosphate indicated that NBCs were 12 times more dense than PBCs. Mean area of cyanophycin granules (N storage) was not significantly different between PBCs and NBCs. In conclusion, Trichodesmium tenue can regulate buoyancy by carbohydrate ballasting similar to that noted in limnetic cyanobacteria. Polyphosphate storage and possibly nitrogen storage products play a significant role in buoyancy regulation. 相似文献
19.
B. A. Lawrence C. Suarez A. DePina Eleanor Click Nancy H. Kolodny M. M. Allen 《Archives of microbiology》1998,169(3):195-200
Two intracellular pools of soluble polyphosphate were identified by in vivo 31P NMR spectroscopy in the cyanobacterium Synechocystis sp. strain PCC 6308. Polyphosphate was present in the cells after growth in sulfur-limited media containing excess phosphate.
The presence of polyphosphate was confirmed by transmission electron microscopy and chemical analysis. 31P NMR spectroscopy of whole cells treated with EDTA revealed two pools of mobile polyphosphate. A downfield shift and narrowing
of part of the broad polyphosphate resonance was observed after EDTA treatment, suggesting that EDTA binds metal ions normally
associated with some of the polyphosphate. Phosphate, but not polyphosphate, leaked out of the cells after this treatment.
Addition of magnesium ions caused the downfield shift in the polyphosphate resonance to move back toward its original value.
These data show that only part of the cation-complexed polyphosphate is accessible to the added EDTA and suggest that there
are two internal fractions of NMR-visible polyphosphate in the cells, only one of which loses its associated cations to EDTA.
Spheroplast formation showed that polyphosphate was not present in the periplasm of the cells.
Received: 3 July 1997 / Accepted: 26 September 1997 相似文献