共查询到20条相似文献,搜索用时 15 毫秒
1.
K M Brindle 《Biochemistry》1988,27(16):6187-6196
31P NMR magnetization-transfer measurements were used to measure flux between inorganic phosphate and ATP in the reactions catalyzed by phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase in anaerobic cells of the yeast Saccharomyces cerevisiae. Flux between ATP and Pi and glucose consumption and ethanol production were measured in cells expressing different levels of phosphoglycerate kinase activity. Overexpression of the enzyme was obtained by transforming the cells with a multicopy plasmid containing the phosphoglycerate kinase coding sequence and portions of the promoter element. Fluxes were also measured in cells in which the glyceraldehyde-3-phosphate dehydrogenase activity had been lowered by limited incubation with iodoacetate. These measurements showed that both enzymes have low flux control coefficients for glycolysis but that phosphoglycerate kinase has a relatively high flux control coefficient for the ATP----Pi exchange catalyzed by the two enzymes. The Pi----ATP exchange velocities observed in the cell were shown to be similar to those displayed by the isolated enzymes in vitro under conditions designed to mimic those in the cell with respect to the enzyme substrate concentrations. 相似文献
2.
The interaction between 1-anilino-8-naphthalenesulfonate (ANS) and yeast phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) and the use of ANS as a probe for studying the structure and function of phosphoglycerate kinase has been investigated. The interaction has been studied by kinetic methods, equilibrium dialysis, and fluorometric titrations. ANS inhibits the activity of the enzyme. More than one inhibitor site exists. ANS is competitive with MgATP and noncompetitive with 3-phosphoglycerate at the first detected inhibitor binding site. The Ki value is 1-2 mM. Several ANS molecules bind to the enzyme. By fluorometric titrations the first detected site has a dissociation constant that is in the same range as Ki or bigger. When ANS interacts with phosphoglycerate kinase its fluorescence is increased and a blue shift occurs. ANS appears to bind to a strongly hydrophobic site. The fluorescence is sensitive to the addition of substrates. ADP, ATP, or combinations of Mg2+ and nucleotide decreases the fluorescence as does free Mg2+. 3-Phosphoglycerate, on the other hand, increases the fluorescence giving evidence for conformational changes upon 3-phosphoglycerate binding. 相似文献
3.
Measurements of the relaxation rate of water protons (PRR) have been used to study the interaction of yeast phosphoglycerate kinase with the manganous complexes of a number of nucleotides. The results indicate that phosphoglycerate kinase belongs to the same class of enzymes as creatine kinase, adenylate kinase, formyltetrahydrofolate synthetase, and arginine kinase, with maximal binding of metal ion to tne enzyme in the presence of the nucleotide substrate. However, an analysis of titration curves for a number of nucleoside diphosphates (ADP, IDP, GDP) showed that there is a substantial synergism in binding of the metal ion and nucleotide to the enzyme in the ternary complex. The metal-substrate binds to the enzyme approximately two orders of magnitude more tightly than the free nucleotide; Other evidence for an atypical binding scheme for Mn(II)-nucleoside diphosphates was obtained by electron paramagnetic resonance (EPR) studies; the EPR spectrum for the bound Mn(II) in the enzyme-MnADP complex differed substantially from those obtained for other kinases. An identical EPR spectrum is observed with the MnADP complex with the rabbit muscle enzyme as with the yeast enzyme. In contrast, the dissociation constant for the enzyme-MnATP complex is approximately fourfold lower than that for enzyme-ATP, and there are no substantial changes in the electron paramagnetic resonance spectrum of MnATP2- when the complex is bound to phosphoglycerate kinase. A small but significant change in the PRR of water is observed on addition of 3-phosphoglycerate (but not 2-phosphoglycerate) to the MnADP-enzyme complex. However, addition of 3-phosphoglycerate to enzyme-MnADP did not influence the EPR spectrum of the enzyme-bound Mn(II). 相似文献
4.
The well-known soluble kinases are not specific for ATP (1). All these enzymes convert ATP as well as GTP, ITP, CTP, and UTP, although at different rates. The only exception is adenylate kinase (1). However, with this enzyme, a direct determination of ATP in tissue extracts which contain both the di- and mononucleotides is not possible.Phosphoglycerate kinase from various sources is specific for ATP, GTP, and ITP and does not react with the pyrimidine nucleotides (2), Now, however, it was found that phosphoglycerate kinase from the blue alga Spirulina platensis does not convert GTP and ITP. With this enzyme, therefore, it is possible to specifically determine ATP in tissue extracts or in mixtures of nucleotides. In the same test, GTP and ITP can be determined by adding phosphoglycerate kinase from yeast or from other sources (2). 相似文献
5.
The well-known soluble kinases are not specific for ATP (1). All these enzymes convert ATP as well as GTP, ITP, CTP, and UTP, although at different rates. The only exception is adenylate kinase (1). However, with this enzyme, a direct determination of ATP in tissue extracts which contain both the di- and mononucleotides is not possible.Phosphoglycerate kinase from various sources is specific for ATP, GTP, and ITP and does not react with the pyrimidine nucleotides (2), Now, however, it was found that phosphoglycerate kinase from the blue alga Spirulina platensis does not convert GTP and ITP. With this enzyme, therefore, it is possible to specifically determine ATP in tissue extracts or in mixtures of nucleotides. In the same test, GTP and ITP can be determined by adding phosphoglycerate kinase from yeast or from other sources (2). 相似文献
6.
A single SH-group of phosphoglycerate kinase from yeast was modified by mercury-containing spin label. The saturation curves of ESR spectra of the spin-labeled enzyme were studied. The paramagnetic ions of Mn2+ bound to the centre of ion nonspecific binding or active centre in the complex with ATP can influence the saturation of the spin-labeled enzyme. The saturation curves of the ESR signal of the spin-labeled enzyme in the presence of paramagnetic complex of CrATP were studied. It has been demonstrated that the second nonspecific centre of ATP binding is located at the active site of the enzyme (3-phosphoglycerate binding centre). 相似文献
7.
(1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10--20 min at 79 degrees C. (2) Catalytic properties such as pH optimum (pH 6--8.5), kinetic parameters (Km=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. (3) The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0 The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% alpha-helix and 11% pleated sheet from the circular dichroic spectrum of the enzyme protein. (4) The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase. 相似文献
8.
Exchange-inert beta, gamma-bidentate Cr(H2O)x(NH3)y ATP complexes inactivate yeast phosphoglycerate kinase (PGK) by forming a coordination complex at the enzyme active site. The observed inactivation rates ranged from 0.019 min-1 to 0.118 min-1 for Cr(NH3)4ATP and Cr(H2O)4ATP, respectively. Incorporation of one mol of Cr-ATP to the enzyme was sufficient for complete inactivation of the enzyme. The presence of Mg-ATP protected the enzyme against inactivation by Cr-ATP. The other substrate 3-phosphoglycerate (3-PGA), when present, reduced the observed inactivation rates. The reduction of the k(obs) by 3-PGA was proportional to the number of NH3 ligands present in the coordination sphere of Cr3+ in the Cr-ATP complex, suggesting that in the ternary enzyme-Cr-ATP-3-PGA complex 3-PGA may be coordinated to the metal ion. When the effector sulfate ion was present, the presence of 3-PGA did not cause any further effects on the observed inactivation rates. This suggests that bound substrates are in a different arrangement at the active site when sulfate is present and therefore 3-PGA may not need to displace a ligand from Cr3+. Additionally, PGK exhibited a stereoselectivity for the binding of Cr(H2O)4ATP. delta diastereomer of Cr(H2O)4ATP yielded an order of magnitude smaller Ki value compared to the value observed with the lambda isomer. The recovery of enzyme activity was observed over a period of a few hours upon removal of excess Cr-ATP. The presence of substrates and/or effector ion sulfate did not alter the observed reactivation rate. There was no difference in the reactivation rates of the enzyme which was inactivated with Cr(H2O)4ATP or Cr(NH3)4ATP with and without 3-PGA. Increasing the ligand exchange rates of Cr3+ of Cr-ATP by increasing the pH value of the recovery medium from 5.9 to 6.8 increased the rate of recovery by a factor of 8. The pH dependence of the reactivation indicated that one hydroxyl group is involved in the recovery of the enzyme activity in enzyme CrATP and enzyme.CrATP.3-PGA complexes. 相似文献
9.
P. D. SWANSON 《Journal of neurochemistry》1968,15(10):1159-1167
—Microsomal fractions prepared from guinea pig cerebral cortex manifested ADP-ATP exchange activity, 40–99 per cent of which was extractable by dilute salt solutions. All of the (Na+, K+)-ATPase activity remained in the particulate material. The unextracted ADP-ATP exchange activity was stimulated six to seven fold by a non-ionic detergent (Lubrol W). When pre-extracted microsomes were sedimented in a sucrose density gradient, the ADP-ATP exchange activity was more widely distributed than (Na+, K+)-ATPase or adenylate kinase activities. The ADP-ATP exchange activity of microsomes extracted with NaI was stimulated by Na+ ions when the Mg2+ concentration in the reaction mixture was low (0·2 mm ). The Na+ stimulation of exchange activity was more variable than was the stimulation of phosphate formation by Na+ plus K+. The Na+-stimulated ADP-ATP exchange reaction of extracted microsomes may be a component of the (Na+, K+)-ATPase system, which has not been freed from adenylate kinase or possibly other contributing enzyme systems. 相似文献
10.
Kinetic studies of adenosine kinase from L1210 cells: a model enzyme with a two-site ping-pong mechanism 总被引:1,自引:0,他引:1
Purified adenosine kinase from L1210 cells displayed substrate inhibition by high concentrations of adenosine (Ado), ATP, and MgCl2. When incubated with ATP and MgCl2, the enzyme was phosphorylated, and the phosphorylated kinase transferred phosphate to adenosine in the absence of ATP and MgCl2. Substrate binding, isotope exchange, and kinetic studies suggested that the enzyme catalyzes the reaction by means of a two-site ping-pong mechanism with the phosphorylated enzyme as an obligatory intermediate. Among many possible pathways within this mechanism probably a random-bi ordered-bi route is the preferred sequence in which the two substrates, adenosine and MgATP, bind in a random order to form the ternary complex MgATP . E . Ado followed by the sequential dissociation of MgADP and AMP. Dissociation constants of various enzyme-substrate and enzyme-product complexes and the first-order rate constant of the rate-limiting step were estimated. 相似文献
11.
Human phosphoglycerate kinase has been totally purified from leukemic granulocytes by double 'affinity elution' with ATP and 3-phosphoglycerate. This purification procedure allowed to obtain 19 mg of protein, specific activity of which was 400 IU/mg i.e. a 105-fold purification and an overall yield of 47%. Purified enzyme was homogenous when tested by acrylamide sodium dodecyl sulfate electrophoresis and immunodiffusion. Specific antibodies raised in rabbits totally inactivated phosphoglycerate kinase of crude extracts as well as of the purified preparation. The molecular specific activity (i.e. the ratio enzyme activity/immunological reactivity) was identical in leukocytes, platelets, erythrocytes and was identical in these cells to the value found for the totally purified phosphoglycerate kinase. 相似文献
12.
Reversible thermal denaturation of phosphoglycerate kinases (E.C. 2.7.2.3) from an extremely thermophilic bacterium Thermus thermophilus and from yeast were studied by measuring their circular dichroism and fluorescence intensity. The thermal denaturation in the presence of guanidine hydrochloride was completely reversible. The thermodynamic parameters for the reaction were calculated based on a two-state mechanism. The free energy changes in denaturation at 25 °C in the absence of denaturant were estimated to be 11.87 ± 0.21 kcal/mol for T. thermophilus phosphoglycerate kinase and 5.33 ± 0.13 kcal/mol for that of yeast. It was found that the van't Hoff plot of the equilibrium constant for the denaturation reaction was almost independent of temperature in the temperature range 0 to 60 °C for T. thermophilus phosphoglycerate kinase, while that of yeast phosphoglycerate kinase was strongly temperature-dependent as reported for other thermolabile proteins. The enthalpy change in denaturation varies from 0.03 to 6.2 kcal/mol (0 to 60 °C) for T. thermophilus phosphoglycerate kinase and from ?27 to 31 kcal/mol (10 to 35 °C) for yeast enzyme. The entropy change in denaturation varies from ?3.9 to 21 entropy units for T. thermophilus phosphoglycerate kinase and ?96 to 104 entropys unit (10 to 35 °C) for yeast enzyme. The heat capacity change in denaturation is between 1.4 and 63 cal/deg. mol for the thermophile enzyme and between 1530 and 1750 cal/deg. mol for yeast enzyme at 20 °C. The observations that the enthalpy changes as well as the heat capacity changes in denaturation of the thermophilic enzyme were negligibly small suggest an explanation for the unusual stability to heat of T. thermophilus phosphoglycerate kinase.We also propose three possible mechanisms for the thermostability of proteins in general. 相似文献
13.
Cibacron Blue 3G-A (CB3G-A, I) was investigated as a ternary complex analogue of lactate dehydrogenase and phosphoglycerate kinase as had been suggested earlier (Stellwagen, E. (1977) Accts. Chem. Res. 10, 92-98). CB3G-A and Procion Brilliant Blue (PBB, II), a structural isomer of the Cibacron dye without the sulfonated benzoyl moiety, were attached covalently to Sepharose CL-6B. The two enzymes were adsorbed to the columns and then eluted by substrates in various combinations. CB3G-A and PBB interact similarly with the two enzymes in spite of the structural differences between the dyes indicating that the specific structure of CB3G-A is not a ternary complex analogue. Inhibition studies of phosphoglycerate kinase by CB3G-A suggest that 2 molecules of dye bind per monomer and are consistent with multiple substrate binding sites. It is suggested that the kinetic mechanism of phosphoglycerate kinase is best described as steady state random. 相似文献
14.
1. K(+) did not affect the Mg(2+)-dependent transphosphorylation but markedly increased the Na(+)-stimulated ADP-ATP exchange rate mediated by a microsomal fraction from guinea-pig kidney. 2. Rb(+), Cs(+), NH(4) (+) and Li(+) were equally effective in stimulating the Na(+)-dependent ADP-ATP exchange activity. 3. Treatment of the microsomal fraction with N-ethylmaleimide or increased concentrations of Mg(2+) prevented stimulation of the Na(+)-dependent exchange reaction by K(+). 4. Ouabain (2.5mum) inhibited ATP hydrolysis by 33% but did not decrease the K(+)-stimulated Na(+)-dependent ADP-ATP exchange rate. 5. A possible mechanism for stimulation of exchange activity by K(+) is discussed. 相似文献
15.
A steady-state kinetic analysis of the fructose 1,6-bisphosphate-activated pyruvate kinase from Carcinus maenas hepatopancreas.
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1. An investigation of the reaction mechanism of the fructose 1,6-bisphosphate-activated pyruvate kinase isolated from the hepatopancreas of the crab Carcinus maenas was conducted. The enzyme was assayed in the presence of 500 microns-fructose 1,6-bisphosphate, 75 mM-KCl and 8 mM-Mg2+free at 25 degrees C. The results are consistent with a rapid-equilibrium random mechanism. 2. Evidence is presented that suggests the formation of two mixed-substrate-product dead-end complexes, enzyme-ADP-pyruvate and enzyme-ADP-ATP. 3. Competitive substrate inhibition was observed for both substrates, ADP and phosphoenolpyruvate, suggesting the formation of the complexes enzyme-ADP-ADP and enzyme-phosphoenolpyruvate-phosphoenolpyruvate in the suggested mechanism. 4. Data from the ATP product-inhibition studies indicate the formation of the complex enzyme-ATP-ATP. This suggests that in the reverse reaction ATP also will show substrate inhibition. 5. The presence of a saturating concentration of fructose 1,6-bisphosphate does not cause full activation of the purified preparations of the enzyme. 6. Pyruvate kinase activity in the supernatant of a hepatopancreas homogenate was completely activated by fructose 1,6-bisphosphate, suggesting that the binding of this ligand to the purified pyruvate kinase was impaired. 相似文献
16.
The enzyme helicase III catalyzes ATP-dependent unwinding of double-stranded DNA (Yarranto, G. T., Das, R. H., and Gefter, M. L. (1979) J. Biol. Chem. 254, 11997-12001). The free enzyme is able to bind to double- and single-stranded DNA. In the presence of ATP the enzyme can bind single- but not double-stranded DNA. The enzyme catalyzes an ADP-ATP exchange reaction in the absence of DNA. It is suggested that there is an enzyme.phosphate complex that discriminates between the two forms of DNA. These results are discussed in relation to a model that accounts for catalytic unwinding of DNA coupled to ATP hydrolysis. 相似文献
17.
C A Pickover D B McKay D M Engelman T A Steitz 《The Journal of biological chemistry》1979,254(22):11323-11329
Using small angle x-ray scattering from solutions of yeast phosphoglycerate kinase, we have measured the radius of gyration of the enzyme both in the presence and in the abscence of ligands. We find that the radius of gyration decreases by 1.09 +/- 0.34 A upon binding both substrates MgATP and 3-phosphoglycerate to form the ternary complex. Smaller decreases, at the limit of the precision of the measurement, were found for the separate binding of MgATP (0.30 +/- 0.50 A). Using computer modeling, it has been estimated that a substrate-induced cleft closure in phosphoglycerate kinase resulting from one lobe rotating 8-14 degrees relative to the other lobe lobe is consistent with this observed change in radius of gyration. We suggest, therefore, that the conformational change that results in the smaller radius of gyration for the ternary complex is a hinge motion of the two lobes which produces a closing of the cleft between the two lobes. The apparent similarity of the ligand-induced change in phosphoglycerate kinase to the cleft closure in hexokinase suggests that this kind of conformational change may prove to be a rather general kinase phenomenon (Bennett, W.S., and Steitz T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852; Anderson, C.M., Zucker, F.H., and Steitz, T.A. (1979) Science 204, 375-380). 相似文献
18.
The single thiol of yeast phosphoglycerate kinase was labelled with the chromophoric sulfhydryl reagent, 2-chloromercuri-4-nitrophenol. Sequential additions of individual anions to this modified enzyme brought about a decrease in absorbance at 410 nm that reflected the degree of saturation of the enzyme with anion. The binding curves were analyzed to determine the dissociation constants of a number of anions with charges varying from--1 to--4.1. A linear relationship was found between the charge of the anion and the negative logarithm of the dissociation constant for the labelled enzyme-anion complex. The highly charged anions, such as ATP, bound more tightly than did anions with less charge, such as Cl-. The average number of binding sites for those anions for which accurate results could be obtained was 1.06 mol per 47000 g of enzyme. Several lines of evidence suggested that titration of the active center was not being monitored. Anions bound to phosphoglycerate kinase decreased the rate of reaction between the enzyme thiol and 5,5'-dithiobis(2-nitrobenzoic acid). The relationship between the degree of saturation of the anion binding site and the reaction rate constant was used to calculate the dissociation constant between anion and enzyme. Dissociation constants determined in this manner were in good agreement with those determined by titration of the enzyme-mercurial complex. 相似文献
19.
Human nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) catalyzes the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PP i) from nicotinamide (NAM) and alpha- d-5-phosphoribosyl-1-pyrophosphate (PRPP). NAMPT, by capturing the energy provided by its facultative ATPase activity, allows the production of NMN at product:substrate ratios thermodynamically forbidden in the absence of ATP. With ATP hydrolysis coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, substrate affinity dramatically increases ( K m (NAM) from 855 to 5 nM), and the K eq shifts -2.1 kcal/mol toward NMN formation. ADP-ATP isotopic exchange experiments support the formation of a high-energy phosphorylated intermediate (phospho-H247) as the mechanism for altered catalytic efficiency during ATP hydrolysis. NAMPT captures only a small portion of the energy generated by ATP hydrolysis to shift the dynamic chemical equilibrium. Although the weak energetic coupling of ATP hydrolysis appears to be a nonoptimized enzymatic function, closer analysis of this remarkable protein reveals an enzyme designed to capture NAM with high efficiency at the expense of ATP hydrolysis. NMN is a rate-limiting precursor for recycling to the essential regulatory cofactor, nicotinamide adenine dinucleotide (NAD (+)). NMN synthesis by NAMPT is powerfully inhibited by both NAD (+) ( K i = 0.14 muM) and NADH ( K i = 0.22 muM), an apparent regulatory feedback mechanism. 相似文献
20.
Blue dextran or Cibacron Blue F3GA has been shown to inhibit yeast phosphoglycerate kinase [EC 2.7.2.3] competitively with respect to ATP (Thompson et al. (1975) Proc. Natl. Acad. Sci. U.S. 72, 663--667; Beissner and Rudolph (1979) J. Biol. Chem. 254, 6273--6277). However, we have found that phosphoglycerate kinase of Lactobacillus plantarum was inhibited by Cibacron Blue F3GA, the blue chromophore of blue dextran, noncompetitively with respect to ATP, but competitively with respect to 3-phosphoglycerate. Further inhibition studies with Cibacron Blue F3GA suggest that one molecule of the dye was bound per molecule of phosphoglycerate kinase at a saturated level of either substrate, but two molecules of the dye were bound per molecule of the kinase with an unsaturated level of either substrate used as a fixed substrate. Furthermore, phosphoglycerate mutase [EC 2.7.5.3] of Leuconostoc dextranicum was also inhibited by Cibacron Blue F3GA competitively with respect to 3-phosphoglycerate and noncompetitively with respect to 2,3-bisphosphoglycerate. These results suggest that the 3-phosphoglycerate-binding site on both phosphoglycerate kinase and phosphoglycerate mutase can interact with Cibacron Blue F3GA. 相似文献