Article

Complex Formation between 3‐Phospho‐d‐glyceric Acid and Divalent Metal Ions

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Abstract

Stoichiometric dissociation constants of complexes between the fully deprotonated 3-phospho-d-glyceric acid and Mg2+, Mn2+, Ca2+, Co2+, Zn2+, and Ni2+, respectively, were determined in 0.25 M NaCl at 25°C. Potentiometric titrations of the ligand with these divalent metal ions were performed over a pH range close to the pK for the final deprotonation step of the ligand. The observed pH changes were utilized to estimate the dissociation constants; both algebraic and graphical techniques were employed in the estimations. Under the experimental conditions 1:1 complexes were the predominant products.

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... These calculations also corrected for the amount of metal chelated by 3PGA at different pHs. 3PGA can exist in different protonated states depending on the pH, but the free form (3PGA 3-) is the only one that significantly binds metal ions (Larsson-Raznikiewicz 1972). The effective concentration of this species was determined at each working pH using published acid dissociation constants (Larsson-Raznikiewicz 1972). ...
... 3PGA can exist in different protonated states depending on the pH, but the free form (3PGA 3-) is the only one that significantly binds metal ions (Larsson-Raznikiewicz 1972). The effective concentration of this species was determined at each working pH using published acid dissociation constants (Larsson-Raznikiewicz 1972). Thereafter, the concentration of free metal ion was calculated using published dissociation constants for metal ion -3PGA chelates; although these latter values were determined at pHs 6 and 6.5, they are not significantly different at alkaline pHs (Larsson-Raznikiewicz 1972). ...
... The effective concentration of this species was determined at each working pH using published acid dissociation constants (Larsson-Raznikiewicz 1972). Thereafter, the concentration of free metal ion was calculated using published dissociation constants for metal ion -3PGA chelates; although these latter values were determined at pHs 6 and 6.5, they are not significantly different at alkaline pHs (Larsson-Raznikiewicz 1972). The reaction was allowed to proceed at 26°C for various lengths of time, and after stopping the reaction and destroying Pgm with trichloroacetic acid and N-ethylmaleimide, the amount of 2PGA formed was measured in the second step of the assay as described (Kuhn et al. 1993). ...
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The enzymatic activity of phosphoglycerate mutase (Pgm) from three gram-positive endospore-forming bacteria (Bacillus subtilis, Clostridium perfringens, and Sporosarcina ureae) requires Mn2+ and is very sensitive to pH; at low concentrations of Mn2+, a pH change from 8 to 6 resulted in greater than 30- to 200-fold decreases in the activity of these Pgms. However, Pgm deactivation at pH 6 was reversed by shifting the enzyme to pH 7 or 8. Free Mn2+ was not directly involved in Pgm catalysis, although enzyme-bound Mn2+ may be involved. The rate of catalysis by Mn2+-containing Pgm was also slightly pH dependent, although the Km for 3-phosphoglyceric acid appeared to be the same at pH 6, 7, and 8. These findings suggest that Mn2+ binds to catalytically inactive Pgm and converts it to a catalytically competent form, and further, that pH influences the efficiency with which the enzyme binds Mn2+. The extreme pH sensitivity of the Mn2+-dependent Pgms supports a model in which this enzyme is inhibited during sporulation by acidification of the forespore, thus allowing accumulation of the spore's large depot of 3-phospho-glyceric acid. The activity of Pgm from two closely related gram-positive bacteria that do not form spores (Planococcus citreus and Staphylococcus saprophyticus) also requires Mn2+ and is pH sensitive. In contrast, the Pgm activities from two more distantly related non-endospore-forming gram-positive bacteria (Micrococcus luteus and Streptomyces coelicolor) are neither dependent on metal ions nor particularly sensitive to pH.Key words: Bacillus, Clostridium, Mn2+, phosphoglycerate mutase, sporulation.
... The barium salt of 3-P-glycerate was dissolved with one equivalent of and the BaS04 precipitate removed by centrifugation. The solution was brought to pH 7.5 using triethanolamine, and was standardized enzymatically using phosphoglycerate kinase and glyceraldehyde-phosphate dehydrogenase, with 10 mM hydrazine present to ensure completion of the reaction [19]. This solution was also adjusted to 100 mM. ...
... The constant (0.025 mM) was close to the value obtained by Blair [20]. For Mg-3-P-glycerate, the value was extrapolated from several available results, none of which were carried out in exactly the present conditions [ 19,221. Should any errors be introduced due to use of incorrect dissociation constants for these complexes, they will be of a quantitative rather than qualitative nature, mainly in the experiment where a high MgL+ concentration was used. ...
Article
1. A re-investigation of the kinetics of yeast phosphoglycerate kinase in the direction of 1,3-bisphosphoglycerate formation has been carried out, covering a 1000-fold range in substrate concentrations. A variety of improved spectrophotometric and fluorimetric assay procedures have been used. 2. Kinetic plots proved to be non-linear for each variable substrate. A variety of checks have been carried out to show that this is not due to artifacts in the assay procedures or heterogeneity of the enzyme preparation. 3. The effects of a variety of salts on the activity of the enzyme have been examined. Most salts, especially those with multivalent anions, can cause activation of the enzyme, but inhibit at high concentration. 4. The salt effect is shown to be principally due to anions rather than cations, and not to ionic strength changes. Sulphate, as one of the most effective anions has been used in most comparisons. 5. Salt activation is steepest when the substrate concentrations are low; maximum activation has been about 5-fold with 0.2 mM MgATP and 0.2 mM 3-phosphoglycerate. Inhibition at the higher salt concentrations is strongest at the same substrate concentrations as when activation is steepest, indicating a link between the two effects. 6. The presence of 20 mM or more Na2SO4 converted non-linear kinetic plots to linear ones. A study of the kinetics in the presence of 40 mM Na2SO4 was interpreted in terms of a random sequential binding mechanism, with sulphate acting as a competitive inhibitor. 7. Possible explanations for these anomalous results are discussed in terms of several mechanisms which have been shown to apply in other systems.
... Ionization constants for the following acids were obtained from the CRC Handbook of Biochemistry [12] : citric, oxalic, succinic, phosphoric, glycerol 2phosphoric, acetic, and sulfuric ; those for ATP, ADP, and AMP were from Perrin [13]. Ionization constants for 3-phosphoglyceric acid, 2,3-bisphosphoglyceric acid, and pyrophosphoric acid were as reported by Larsson-Rainikiewicz [14], Yap and Saroff [I51 and Moe and Wiest [16], respectively. The anion charge at pH 8.0 was calculated by use of the Henderson-Hasselbach equation. ...
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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.
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Detailed kinetic studies, including initial velocity, product inhibition, and substrate analog inhibition measurements, have been carried out on the reverse reaction catalyzed by human erythrocyte phosphoglycerate kinase with MgATP2- as the phosphoryl group donor. The results are consistent with the reaction conforming to a rapid equilibrium random mechanism. Substantially similar results were obtained for other activating metal ions (manganese, calcium, and cobalt) and other nucleoside triphosphates (ATP, GTP). An ADP ATP exchange reaction was invariably associated with the purified enzyme but definitive evidence that it was an intrinsic property of the enzyme was not obtained.
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The enzymatic activity of phosphoglycerate mutase (Pgm) from three gram-positive endospore-forming bacteria (Bacillus subtilis, Clostridium perfringens, and Sporosarcina ureae) requires Mn2+ and is very sensitive to pH; at low concentrations of Mn2+, a pH change from 8 to 6 resulted in greater than 30- to 200-fold decreases in the activity of these Pgms. However, Pgm deactivation at pH 6 was reversed by shifting the enzyme to pH 7 or 8. Free Mn2+ was not directly involved in Pgm catalysis, although enzyme-bound Mn2+ may be involved. The rate of catalysis by Mn(2+)-containing Pgm was also slightly pH dependent, although the Km for 3-phosphoglyceric acid appeared to be the same at pH 6, 7, and 8. These findings suggest that Mn2+ binds to catalytically inactive Pgm and converts it to a catalytically competent form, and further, that pH influences the efficiency with which the enzyme binds Mn2+. The extreme pH sensitivity of the Mn(2+)-dependent Pgms supports a model in which this enzyme is inhibited during sporulation by acidification of the forespore, thus allowing accumulation of the spore's large depot of 3-phosphoglyceric acid. The activity of Pgm from two closely related gram-positive bacteria that do not form spores (Planococcus citreus and Staphylococcus saprophyticus) also requires Mn2+ and is pH sensitive. In contrast, the Pgm activities from two more distantly related non-endospore-forming gram-positive bacteria (Micrococcus luteus and Streptomyces coelicolor) are neither dependent on metal ions nor particularly sensitive to pH.
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A kinetic study of the effects of SO4(2-) in the activity of phosphoglycerate kinase (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) is presented. SO4(2-) behaves both as an activator and inhibitor of the reaction. Activation does not appear to affect binding of one or the other of the two substrates to the catalytic centre. As an inhibitor SO4(2-) competes with both the substrates. Thus, each substrate can constrict SO4(2-) from the inhibitor binding site, probably the catalytic centre. Under these conditions activation becomes more and more evident. There appear to exist at least two SO4(2-) binding sites, one which earlier has been defined as an anion binding site, and a second, being the catalytic centre. The former seems to have a higher affinity for SO4(2-) than the latter.
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Metal ion binding to yeast phosphoglycerate kinase was monitored with two systems that were sensitive to the conformation of the protein. One system used the enzyme labelled with the sulfhydryl reporter group 2-chloromercuri-4-nitrophenol. Titrations of this complex with Mg2+, Mn2+, Co2+, Ca2+ or Cd2+ increased the absorbance of the bound chromophore. Dissociation constants for these metal ions to the complex were determined from Scatchard plots and ranged from 8 μM to 43 μM. A single class of binding site per 47000 g of enzyme was indicated. In contrast, two classes of Zn2+ binding sites were demonstrated and both appeared to be distinct from the other class of metal ion binding sites. A second system involved the reaction between native phosphoglycerate kinase and 5,5′-dithio-bis(2-nitrobenzoic acid). The rate constant for this reaction increased in the presence of each of the divalent metal ions examined. Analysis of the data gave dissociation constants which agreed with those determined from the mercurial system. Further, the same two classes of Zn2+ binding sites were apparent. The affinity of the native enzyme for anions as previously reported by us [Eur. J. Biochem. 85, 345–350 (1978)] was not influenced by occupancy of the metal ion binding site with Mg2+ or Ca2+, or by saturation of one of the two classes of Zn2+ binding sites. Anion binding did not alter the affinity of the enzyme for Mg2+ or Ca2+, nor did it significantly weaken the binding of Zn2+ to one of its sites.
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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.
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The kinetic properties of rat liver phosphoglycerate kinase were investigated in the forward direction of the reaction (utilization of ADP). The kinetic studies were performed in an assay system using combined hexokinase/glucose-6-phosphate dehydrogenase as an ATP trap. The Km values for Mg ADP1- and 1,3-diphospho-D-glycerate were approximately 0.11 and 0.006 mM, respectively. Reciprocal plots of 1/v versus 1/ (Mg ADP1-) at different fixed concentrations of 1,3-diphospho-D-glycerate and 1/v versus 1/ (1,3-diphospho-D-glycerate) at different fixed concentrations of Mg ADP1- were apparently parallel. However, product inhibition studies (3-phospho-D-glycerate), dead-end inhibition studies (2,3-diphospho-D-glycerate), and adenosine and AMP inhibition patterns yielded results consistent with a rapid equilibrium random mechanism in which the binding of one substrate greatly decreases the affinity of the enzyme for the second substrate. Existence of two sites for 3-phospho-D-glycerate is suggested.
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Equilibrium studies on ATP4− and 3-P-glycerate binding to phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) have been performed. The results show that the enzyme contains two binding sites for both ligands, as was earlier suggested to explain some of the kinetic results. As long as structural information is lacking analysis of binding data can be analyzed if assumptions are made whether the sites are equivalent or nonequivalent, and whether ligation of these sites is mutually dependent or not. To understand the experimental results explicit expressions were derived for the limiting slopes and intercepts of Klotz and Scatchard plots, respectively, in terms of the intrinsic affinity constants valid under the separate assumptions made. The algebraic expressions for the limiting slopes were analyzed in order to ascertain the relationship between the form of these graphical plots, the prevailing types of binding site, and the intrinsic affinity constants. The results show that in case the equivalence of the binding sites is questionable it is necessary to be careful when the type of interaction is being determined. In certain cases it might be difficult to distinguish between positive and negative interaction. Sites that appear to be equivalent and independent could equally well be nonequivalent and dependent.
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Yeast phosphoglycerate kinase is irreversibly inactivated upon incubation with 5'-[p-(fluorosulfonyl)-benzoyl]-1-N6-ethenoadenosine (5'-FSB epsilon A), an analogue to the nucleotide substrate. Marked protection against inactivation occurs with MgATP, ATP, MgADP, ADP, and 3-phosphoglycerate, suggesting that a part of the catalytic center is modified. The time dependence of the inactivation is characterized by a nonlinear kinetic profile. Curve fitting of various models for ligand binding to the enzyme suggested a two-site model. Modification of one of the sites appears to protect the catalytically essential site from modification. Stoichiometric studies show that the relationship between moles of 5'-FSB epsilon A incorporated per mole of enzyme and the residual enzymatic activity also shows nonlinear behavior. An extrapolated value of 1.5 mol of bound label/mol of enzyme corresponds to complete inactivation. The apparent overall pseudo first-order rate constant for the reaction between phosphoglycerate kinase and 5'-FSB epsilon A, as well as the separate rate constants for the modification, exhibit saturation behavior with respect to the concentration of 5'-FSB epsilon A, indicative of a rapid reversible binding of the reagent to the enzyme prior to modification.
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A database of thermodynamic properties is developed, which extends a previous database of glycolysis and tricarboxylic acid cycle by adding the reactions of the pentose phosphate pathway. The raw data and documented estimations of solution properties are made electronically available. The database is determined by estimation of a set of parameters representing species-level free energies of formation. The resulting calculations provide thermodynamic and network-based estimates of thermodynamic properties for six reactions of the pentose phosphate pathway for which estimates are not available in the preexisting literature. Optimized results are made available in ThermoML format. Because calculations depend on estimated hydrogen and metal cation dissociation constants, an uncertainty and sensitivity analysis is performed, revealing 23 critical dissociation constants to which the computed thermodynamic properties are particularly sensitive. Database URL: http://www.biocoda.org/thermo
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Activation of ribulose-1,5-bisphosphate carboxylase by CO2 and Mg2+ is slow and reversible. At subsaturating concentrations of CO2 and Mg2+, positive effectors increase and negative effectors decrease the amount of active enzyme at equilibrium. Preequilibrium experiments indicated that both positive and negative effectors inhibit the rates of enzyme activation and deactivation. Greater than 99% inhibition of the activation and deactivation rates was observed at high effector concentrations, indicating that the binding and release of the activators CO2 and Mg2+ occur only with effector-free enzyme. The deactivation rate Ki values for the negative effector ribose 5-phosphate and the positive effectors inorganic phosphate, fructose 1,6-bisphosphate, and 6-phosphogluconate were smaller than the corresponding activation rate Ki values by factors of 2, 6, 25, and 670, respectively. Thus, phosphorylated effectors impede deactivation more than activation. Equilibrium binding studies indicated that the active and inactive enzyme forms have similar affinities for the positive effectors inorganic phosphate (KD = 650 μM) and fructose bisphosphate (KD = 11 μM). The positive effector 3-phosphoglycerate was bound with greater affinity by the inactive enzyme (KD = 25 μM) than by the active enzyme (KD = 76 μM). Thus, preferential binding of positive effectors to the active enzyme form is not responsible for the enhancement of enzyme activation at equilibrium. The promotion of activation by positive effectors is mediated by altering the relative rates of activation and deactivation to favor active enzyme. Equilibrium binding studies indicated that the inactive enzyme had a much greater affinity for ribose 5-phosphate (KD = 42 μM) than did the active enzyme (KD = 480 μM). Preferential binding of this negative effector to inactive enzyme exceeds its stabilizing effect on the active enzyme form and thus causes an overall reduction of activation at equilibrium.
A kinetic study for understanding how the phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) reaction discriminates between MgATP2− and ATP4− is presented. The results show that in contrast to MgATP2-, ATP4− is competitive with 3-phospho-d-glycerate. ATP4− binds to the free enzyme as an inhibitor. When binding to the enzyme-MgATP2−-(3-phospho-d-glycerate) complex, ATP4− acts as an activator.
Article
Phosphoglycerate kinase (MgATP 3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) has been isolated from rat liver with a purification ratio of 960 and a specific activity of 300 IU/mg of protein. The purity of the enzyme preparations was estimated by polyacrylamide gel electrophoresis. The molecular weight, determined by gel filtration is 42 000. The « subunitsize of phosphoglycerate kinase as determined by sodium dodecyl sulfate gel electrophoresis is 46 000, indicating that the enzyme is monomeric. The rate of the enzyme reaction as a function of the concentration of d-3-phosphoglycerate indicated the usual Michaelis Menten relationship. The rate of the enzyme reaction as a function of the concentration of MgATP2− did not fit the usual Michaelis Menten relationship : two distinct regions can be fitted with different straight lines and suggest the presence of two sites for the Mg ATP2−. This hypothesis seems to be confirmed by the study of the action of the free and complexed nucleotides.
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A reproducible procedure for the large-scale preparation of phosphoglycerate kinase frombaker's yeast is described. This method includes autolysis of dried yeast in 0.75 m ammonia, heat treatment, ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose, Cibacron blue 3 G-A-Sepharose 4B pseudoaffinity chromatography, and Sephadex G-100 gel filtration. Approximately 1.7 g of homogeneous phosphoglycerate kinase can be obtained from 1 kg of air-dried bakers' yeast (yield 52%, specific activity 890 units/mg at 25°C). In a few cases further purification was achieved by reversible salting out on Sepharose CL-4B, hydroxylapatite chromatography, or ATP-Sepharose 4B affinity chromatography. Differences in the preparation of phosphoglycerate kinase from yeast with those from pig liver and pig muscle are discussed, especially concerning the interaction of the three enzymes with the chromophores of Cibacron blue- and dextran blue-Sepharose.
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The blood red cell contains millimolar amounts of 2,3-diphosphoglycerate, yet no metal ion stability constants appear to have been determined. We show that in the free ligand the 2-phospho group is about six times more basic than the 3-phospho group. We have determined the stability constants for the binding of Zn2+ which occurs at about 0.2 mM in the red cell, and of Al3+ which reduces red cell size and inhibits haemoglobin synthesis. At physiological pH both metal ions occur predominantly as MA2 complexes. The free Zn2+ concentration permitted within the red cell by the 2:1 complex is about 0.3 μm. 2,3-Diphosphoglycerate binds Al3+ strongly enough so that at equilibrium there should be comparable concentrations of Al3+ on both sides of the red cell membrane.
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The present study shows that MgATP2- or MnATP2- as substrates of phosphoglycerate kinase can be replaced by CaATP2-, ZnATP2-, CoATP2- or NiATP2-. MnATP2- and NiATP2- are about 90% and 15%, respectively, and the other ATP4--metal ion complexes roughly 70% as good substrates as MgATP2-. No measurable activity was found with Be(II) or Fe(III). The effectiveness of the different substrate species appears to be determined by factors such as the size and polarizing capability of the metal ion as well as of the structure of the relevant metal ion complex and the rate of ligand dissociation processes. Detailed kinetics with Zn(II), Mn(II) and Co(II) showed that: (a) Zn2+ is a strong uncompetitive inhibitor of ZnATP2-, Ki approx. 0.02 mM. (b) Mn2+ is a competitive inhibitor of MnATP2- at concentrations < 0.1 mM, Ki approx. 2.3 mM. This inhibition is dependent on the 3-P-glycerate concentration. At about 1 mM and higher concentrations Mn2+ acts as an uncompetitive inhibitor of MnATP2-. (c) Co2+ is a competitive inhibitor of CoATP2- at about 1 mM and higher concentrations, Ki approx. 3 mM. With CoATP2- as substrate the activity is slightly increased in the presence of free Co2+ and/or free ATP4- at concentrations < 0.5 mM. When the CoATP2- concentration is varied, the activity seems not to become constant at concentrations ten times the apparent Michaelis constant for CoATP2-. Equilibrium dialysis studies on the binding of Co2+ to the enzyme showed that this ion binds more strongly to the enzyme than, for example, Mn2+. This probably explains the differences in activation and inhibition observed with these two ions. The kinetic patterns obtained with the different metal ions indicate that Ni(II) behaves as Co(II), Cd(II) as Zn(II) and Ca(II) as Mg(II), which is somewhat similar in behaviour to Mn(II).
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The binding of Mn++ to 3-phosphoglycerate kinase, and to ADP and ATP, has been studied at pH 7.1 and 38 °. The influence of the concentrations of Mn++ and of the various substrates on the rate of the enzymic reaction has also been investigated at the same conditions. The protein displays only a weak, nonspecific interaction with Mn++. By correlating the kinetic and binding data, it is possible to show that activation does not involve binding of Mn++ to the enzyme but rather the formation of a Mn++-ATP complex. The significance of the results in relation to the general problem of the mechanism of metal-ion activation of enzymes is discussed.
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The binding of Mn++ to 3-phosphoglycerate kinase, and to ADP and ATP, has been studied at pH 7.1 and 38 °. The influence of the concentrations of Mn++ and of the various substrates on the rate of the enzymic reaction has also been investigated at the same conditions. The protein displays only a weak, nonspecific interaction with Mn++. By correlating the kinetic and binding data, it is possible to show that activation does not involve binding of Mn++ to the enzyme but rather the formation of a Mn++-ATP complex. The significance of the results in relation to the general problem of the mechanism of metal-ion activation of enzymes is discussed.
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The effect of the concentration of Mg2+, ATP4- and Mg-ATP2- on the velocity of the phopshoglycerate kinase (ATP:d-3-phosphoglycerate 1-phosphotransferase, EC 2.7.2.3) reaction has been studied. Partly new applications of known kinetic equations are given. Three different kinetic relationships support the concept that Mg-ATP2- is the active substrate. It is also seen that mechanisms in which the formation of enzyme-metal or enzyme-nucleotide complexes are the first steps in the catalytic reaction are very unlikely. ATP4- and Mg2+ both seem to have inhibitory effects only.
Article
Using a colorimetric, a titrimetric and a resin-adsorption method, approximately the same value for the binding constant of Ca2+ to ATP has been determined giving the values 7880, 8380 and 9320, respectively (average 8500). With the last two methods the association constant for MgATP has been obtained as 26700 and 22300, respectively (average 24500). With the titrimetric method the same constant for SrATP was found to be 3940 and for BaATP 2350.
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