Changing kinetic properties of the two-enzyme phosphoglycerate kinase/NADP-linked glyceraldehyde-3-phosphate dehydrogenase couple from pea chloroplasts during photosynthetic induction

The kinetics of the two enzyme phosphoglycerate kinase (EC 2.7.2.3)/NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) couple are negatively cooperative and will also fit a model for two enzymes acting on one substrate. When the chloroplast is illuminated apparent negative cooperativity is reduced; maximal velocity of only one of the two enzymes in the two-enzyme model is increased. Even after light activation the activity of glyceraldehyde-3-phosphate dehydrogenase appears to be too low to support photosynthesis at calculated levels of glycerate-1,3-bisphosphate in isolated chloroplasts (Marques, I.A., Ford, D.M., Muschinek, G. and Anderson, L.E. (1987) Arch. Biochem. Biophys. 252, 458–466). The activity of the coupled reaction is apparently sufficient to support observed rates of CO₂ fixation, which suggests that glycerate-1,3-bisphosphate may be channeled from the kinase to the dehydrogenase in vivo.     

Immobilized glyceraldehyde-3-phosphate dehydrogenase forms a complex with phosphoglycerate kinase

Yeast glyceraldehyde-3-phosphate dehydrogenase (GPDH) covalently attached to CNBr-activated Sepharose 4B was shown to be capable of binding soluble yeast phosphoglycerate kinase (PGK) in the course of incubation in the presence of an excess of 1,3-diphosphoglycerate. The association of the matrix-bound and soluble enzymes also occurred if the kinase was added to a reaction mixture in which the immobilized glyceraldehyde-3-phosphate dehydrogenase, NAD, glyceraldehyde-3-phosphate and Pi had been preincubated. Three kinase molecules were bound per a tetramer of the immobilized dehydrogenase and one molecule per a dimer. An immobilized monomer of glyceraldehyde-3-phosphate dehydrogenase was incapable of binding phosphoglycerate kinase. The matrix-bound bienzyme complexes were stable enough to survive extensive washings with a buffer and could be used repeatedly for activity determinations. Experimental evidence is presented to support the conclusion that 1,3-diphosphoglycerate produced by the kinase bound in a complex can dissociate into solution and be utilized by the dehydrogenase free of phosphoglycerate kinase.     

Combined glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase in catecholamine-stimulated guinea-pig cardiac muscle. Comparison with mass-action ratio of creatine kinase.

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP].[Pi])) was derived from creatine kinase and the free [NAD+]/([NADH].[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]).[Pi]) was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol C3.min-1.g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux approximately 5-14 mumol C3.min-1.g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from approximately 3 to 11 mumol C3.min-1.g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4 microM to greater than or equal to 16 microM. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.     

Evidence that 1,3-bisphosphoglycerate dissociation from phosphoglycerate kinase is an intrinsically rapid reaction step

The steady-state kinetics of 1,3-bisphosphoglycerate formation through the action of phosphoglycerate kinase on 3-phosphoglycerate and ATP have been examined. The results show that initial velocities determined by the standard method of coupling bisphosphoglycerate production to NADH reduction in the presence of glyceraldehyde-3-phosphate dehydrogenase do not differ significantly from those determined in the absence of the latter enzyme. This observation invalidates the proposal that bisphosphoglycerate dissociation from phosphoglycerate kinase is much too slow to account for the high rates of phosphoglycerate turnover observed in the coupled two-enzyme system. The capacity for rapid bisphosphoglycerate production and release is an intrinsic catalytic property of phosphoglycerate kinase that does not require the presence of other enzymes or the involvement of a mechanism of channelized (non-diffusional) transfer of bisphosphoglycerate from the producing enzyme to the consuming one.     

Purification and properties of phosphoglycerate kinase from Thermus thermophilus strain HB8.

(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.     

31P NMR magnetization-transfer measurements of flux between inorganic phosphate and adenosine 5'-triphosphate in yeast cells genetically modified to overproduce phosphoglycerate kinase

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.     

Human granulocyte phosphoglycerate kinase. Purification by double affinity elution and immunological study.

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.     

Yeast glyceraldehyde-3-phosphate dehydrogenase. Evidence that subunit cooperativity in catalysis can be controlled by the formation of a complex with phosphoglycerate kinase

Sepharose-bound tetrameric, dimeric and monomeric forms of yeast glyceraldehyde-3-phosphate dehydrogenase were prepared, as well as immobilized hybrid species containing (by selective oxidation of an active center cysteine residue with H2O2) one inactivated subunit per tetramer or dimer. The catalytic properties of these enzyme forms were compared in the forward reaction (glyceraldehyde-3-phosphate oxidation) and reverse reaction (1,3-bisphosphoglycerate reductive dephosphorylation) under steady-state conditions. In the reaction of glyceraldehyde-3-phosphate oxidation, immobilized monomeric and tetrameric forms exhibited similar specific activities. The hybrid-modified dimer contributed on half of the total activity of a native dimer. The tetramer containing one modified subunit possessed 75% of the activity of an unmodified tetramer. In the reaction of 1,3-bisphosphoglycerate reductive dephosphorylation, the specific activity of the monomeric enzyme species was nearly twice as high as that of the tetramer, suggesting that only one-half of the active centers of the oligomer were acting simultaneously. Subunit cooperativity in catalysis persisted in an isolated dimeric species. The specific activity of a monomer associated with a peroxide-inactivated monomer in a dimer was equal to that of an isolated monomeric species and twice as high as that of a native immobilized dimer. The specific activity of subunits associated with a peroxide-inactivated subunit in a tetramer did not differ from that of a native immobilized tetramer; this indicates that interdimeric interactions are involved in catalytic subunit cooperativity. A complex was formed between the immobilized glyceraldehyde-3-phosphate dehydrogenase and soluble phosphoglycerate kinase. Three monomers of phosphoglycerate kinase were bound per tetramer of the dehydrogenase and one per dimer. Evidence is presented that if the reductive dephosphorylation of 1,3-bisphosphoglycerate proceeds in the phosphoglycerate kinase - glyceraldehyde-3-phosphate dehydrogenase complex, all active sites of the latter enzyme act independently, i.e. subunit cooperativity is abolished.     

Multiple forms of human phosphoglycerate kinase.

Phosphoglycerate kinase was isolated by affinity chromatography from human skeletal muscle and erythrocytes. As in the tissue extracts, the purified enzyme showed in Cellogel electrophoresis one major and two minor bands with phosphoglycerate kinase activity. The multiple forms were separated by chromatography on CM-Sepharose. From the three separated forms, A, B, and C, the latter was not detectable in electrophoresis of tissue extracts or in the purified unresolved phosphoglycerate kinase. The faintest, most anodically migrating form observed in the tissue extracts could not be isolated in pure form by chromatography on CM-Sepharose. The electrophoretic mobility of the phosphoglycerate kinase forms depended strongly on the buffer systems used. The different forms had identical molecular weight, substrate affinity, and heat stability and were inhibited to the same extent by antibody. They could also not be separated by column affinity chromatography. Small differences were found in thiol group content and in the specific activity, the latter being a consequence of diminished free sulfhydryl residues. Exposure to either reductive or oxidative conditions changed the specific activity, but did not result in interconversion among the pure forms. The multiple forms probably arise as a result of epigenetic factors occurring after the primary polypeptide chain has been synthesized.     

A study of phosphoglycerate kinase in human erythrocytes. I. Enzyme isolation, purification and assay.

The enzyme ATP-3-phospho-D-glycerate-1-phosphotransferase (EC 2.7.2.3) (phosphoglycerate kinase) has been isolated from human red cells in crystalline form by a modification of the method of Yoshida and Watanabe (1972) J. Biol. Chem. 247, 440-445). The crystalline enzyme was further purified by electrofocusing using carrier ampholytes (pH 7-9). The isoelectric point of phosphoglycerate kinase was estimated to be 8.75. The specific activity of purified phosphoglycerate kinase from electrofocusing was 2200 units per mg of protein at pH 8.3 (37 degrees C). Enzyme activity was assayed in the forward direction leading from 1,3-diphosphoglycerate to a 3-phosphoglycerate using a fluorimetric procedure for NAD-coupled enzymes for the measurement of the reaction rate at very low substrate concentrations. The auxiliary indicator enzymes were added in excess to yield true initial velocity kinetics, i.e. with no time lag upon addition of substrate (1,3-diphosphoglycerate). This was established theoretically using a mathematical model and confirmed experimentally. Further phosphoglycerate kinase was shown to be the rate-limiting step when the assay conditions were varied.     

Mechanism of 1,3-bisphosphoglycerate transfer from phosphoglycerate kinase to glyceraldehyde-3-phosphate dehydrogenase

1. The kinetics of 1,3-bisphosphoglycerate binding to glyceraldehyde-3-phosphate dehydrogenase have been examined by stopped-flow techniques in the absence and presence of phosphoglycerate kinase, using enzyme concentrations in the range 0.5-40 microM. Rate and equilibrium constant estimates for the interaction of the ligand with the two enzymes are reported. 2. The kinetics of ligand transfer from the binary complex of bisphosphoglycerate and phosphoglycerate kinase to the binary complex of NAD+ and glyceraldehyde-3-phosphate dehydrogenase conform excellently to the predictions of a standard free-diffusion mechanism and exhibit no detectable contributions from a mechanism of direct (channelized) transfer of bisphosphoglycerate between the two enzymes. 3. Previously reported evidence that the binary complex of bisphosphoglycerate and phosphoglycerate kinase may act (in the presence of NADH) as a substrate for glyceraldehyde-3-phosphate dehydrogenase according to Michaelis-Menten kinetics is based on a misinterpretation of the experimental observations that can be attributed to neglect of the autocatalytic effect of NAD+ produced during the reaction. Experiments performed under conditions where the autocatalytic effect of NAD+ is eliminated provide clear evidence that the kinetics of utilization of the kinase-bisphosphoglycerate complex for enzymic NADH reduction are consistent with prior dissociation of the complex according to a free-diffusion mechanism of metabolite transfer and incompatible with a mechanism of direct metabolite transfer. 4. A kinetic argument is presented which renders implausible the very idea that direct metabolite transfer between 'soluble' consecutive enzymes in metabolic pathways may offer any catalytic advantages in comparison to metabolite transfer by free diffusion. A mechanism of direct metabolite transfer seems intuitively attractive only because one tends to disregard the diffusional processes required to bring the consecutive enzymes together and to separate them when the transfer has been completed. Direct metabolite transfer would be expected to be catalytically advantageous only in tightly bound multienzyme complexes showing no kinetically significant tendency to dissociate. 5. It is concluded that mechanisms of direct metabolite transfer have not been convincingly demonstrated to apply, nor are they likely to apply, between 'soluble' consecutive enzymes in metabolic pathways, at least not in the glycolytic sequence of reactions.     

Isolation and properties of the glycolytic enzymes from Zymomonas mobilis. The five enzymes from glyceraldehyde-3-phosphate dehydrogenase through to pyruvate kinase.

The five glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase were each purified from extracts of Zymomonas mobilis cells, by using dye-ligand chromatography as the principal step. Two procedures, producing three and two of the enzymes respectively, are described in detail. Z. mobilis glyceraldehyde-phosphate dehydrogenase was found to be similar in most respects to the enzyme from other sources, except for having a slightly larger subunit size. Phosphoglycerate kinase has properties typical for this enzyme; however, it did not show the sulphate activation effects characteristic of this enzyme from most other sources. Phosphoglycerate mutase is a dimer, partially independent of 2,3-bisphosphoglycerate, and has a high specific activity. Enolase was found to be octameric; otherwise its properties were very similar to those of the yeast enzyme. Pyruvate kinase is unusual in being dimeric, and not requiring K+ for activity. It is not allosterically activated by sugar phosphates, having a high activity in the absence of any effectors. Some quantitative differences in the relative amounts of these enzymes, compared with eukaryotic species, are ascribed to the fact that Z. mobilis utilizes the Entner-Doudoroff pathway rather than the more common Embden-Meyerhoff glycolytic route.     

Product inhibition studies of yeast phosphoglycerate kinase evaluating properties of multiple substrate binding sites.

Product inhibition studies on yeast phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) have been performed with 1,3-P2-glycerate. The results indicate that: 1. The catalytic reaction can be affected via four substrate binding sites, two for MgATP2- and two for 3-P-glycerate. 2. There is one catalytic centre per enzyme molecule. 3. The catalytic reaction primarily occurs at the 'first' or 'high affinity' MgATP2- and 3-P-glycerate binding sites. The 'second' set of sub-sites for these substrates are located in a region for regulation of the catalytic reaction. 4. The products of the reaction, 1,3-P2-glycerate and ADP, are preferentially bound to the regulatory region. 5. MgATP2- and 1,3-P2-glycerate are able to bind simultaneously to this region. When liganded with MgATP2- the apparent Ki value for 1,3-P2-glycerate increases from 3 microM to 20 microM.     

Phylogeny and ontogeny of the phosphoglycerate mutases.--V. Inactivation of phosphoglycerate mutase isozymes by histidine-specific reagents

1. The three isozymes of glycerate-2,3-P2 dependent phosphoglycerate mutase present in tissues of mammals and reptiles were inactivated by both treatment with diethylpyrocarbonate and photooxidation with rose bengal. 2. Inactivation of type M isozyme purified from rabbit muscle was complete when two histidine residues per enzyme subunit were carboethoxylated. Hydroxylamine removed the carboethoxy groups, with partial recovery of the enzymatic activity. The cofactor protected the enzyme against inactivation. 3. The inactivation of rabbit muscle phosphoglycerate mutase by photooxidation with methylene blue and rose bengal was sharply pH dependent. The pH profile of enzyme inactivation followed the titration curve of histidine, suggesting that this amino acid was critical for enzyme activity. Glycerate-2,3-P2 did not protect phosphoglycerate mutase against photoinactivation.     

Activation of Chloroplast NADP-linked Glyceraldehyde-3-Phosphate Dehydrogenase by the Ferredoxin/Thioredoxin System

NADP-glyceraldehyde-3-P dehydrogenase of spinach (Spinacia oleracea) chloroplasts was activated by thioredoxin that was reduced either photochemically with ferredoxin and ferredoxin-thioredoxin reductase or chemically with dithiothreitol. The activation process that was observed with the soluble protein fraction from chloroplasts and with the purified regulatory form of the enzyme was slow relative to the rate of catalysis. The NAD-linked glyceraldehyde-3-P dehydrogenase activity that is also present in chloroplasts and in the purified enzyme preparation was not affected by reduced thioredoxin.

When activated by dithiothreitol-reduced thioredoxin, the regulatory form of NADP-glyceraldehyde-3-P dehydrogenase was partly deactivated by oxidized glutathione. The enzyme activated by photochemically reduced thioredoxin was not appreciably affected by oxidized glutathione. The results suggest that although it resembles other regulatory enzymes in its requirements for light-dependent activation by the ferredoxin/thioredoxin system, NADP-glyceraldehyde-3-P dehydrogenase differs in its mode of deactivation and in its capacity for activation by enzyme effectors independently of thioredoxin.

     

Magnetic resonance studies on manganese-nucleotide complexes of phosphoglycerate kinase.

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).     

Glyceraldehyde-3-Phosphate Dehydrogenase (NADP) from Sinapis alba L: Reversible Association of the Enzyme with a Protein Factor as Controlled by Pyridine Nucleotides in Vitro

Aggregation of glyceraldehyde-3-P dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba seedlings during gel filtration on Sepharose 6B is dependent on the presence of a fraction (“binding fraction”) which can be separated from the enzyme by precipitation with 55% ammonium sulfate. Association of the enzyme with this binding fraction is NAD-dependent whereas NADP⁺ causes release. Dithioerythritol (2 mM) has no influence on these reversible processes.

Binding fractions, partially purified by ammonium sulfate and acetone fractionation, were submitted to dodecylsulfate-polyacrylamide gel electrophoresis. They always contain one or two dominant polypeptides with apparent molecular weights 42,000 and 58,000. The 42,000 polypeptide comigrates during dodecylsulfate electrophoresis with the corresponding subunit of the enzyme. It comprises up to 70% of the total protein in partially purified binding fractions and can be regarded as a major protein in seedling extracts.

The differential transport behavior of glyceraldehyde-3-P dehydrogenase (NADP) on Sephadex G-200 in the presence of NAD⁺ and NADP⁺ can be used as a simple and effective purification procedure. The enzyme isolated in this way has an isoelectric point of about 4.5 and maintains under all tested conditions a heterogeneous subunit composition of at least three different polypeptide chains (apparent molecular weights, 39,000, 42,000, 43,000).

The present data suggest that NAD(P)-controlled aggregation of glyceraldehyde-3-P dehydrogenase (NADP) from Sinapis alba L. is due primarily to enzyme association with a separate binding fraction rather than to enzyme polymerization. It is possible that a major component of this binding fraction, the 42,000 polypeptide, consists of “surplus” nonactive enzyme subunits, which self-associate and interact with the NAD-conformer of the enzyme.

     

Effect of Cibacron Blue F3GA on phosphoglycerate kinase of Lactobacillus plantarum and phosphoglycerate mutase of Leuconostoc dextranicum

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.     

Substrate binding to phosphoglycerate kinase monitored by 1-anilino-8-naphthalenesulfonate

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.     

Reversible thermal unfolding of thermostable phosphoglycerate kinase. Thermostability associated with mean zero enthalpy change.

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.