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PubMed2001

Starch synthesis in transgenic potato tubers with increased 3-phosphoglyceric acid content as a consequence of increased 6-phosphofructokinase activity.

The aim of this work was to test the hypothesis that changes in cytosolic 3-phosphoglyceric acid (3-PGA) content can regulate the rate of starch synthesis in potato (Solanum tuberosum L.) tubers. The amount of 3-PGA was increased by expressing bacterial phosphofructokinase (PFK; EC 2.7.1.11) in transgenic potato tubers. The resultant 3-fold increase in PFK activity was accompanied by an increase in metabolites downstream of PFK, including a 3-fold increase in 3-PGA. There was also a decrease in metabolites upstream of PFK, most notably of glucose-6-phosphate. The increase in 3-PGA did not affect the amount of starch that accumulated in developing tubers, nor its rate of synthesis in tuber discs cut from developing tubers. This suggests that changes in cytosolic 3-PGA may not affect the rate of starch synthesis under all circumstances. We propose that in this case, a decrease in glucose-6-phosphate (which is transported into the amyloplast as a substrate for starch synthesis) may be sufficient to counteract the effect of increased 3-PGA.

Carbon Radioisotopes↗
PubMed1983

The arsonomethyl group as an analogue of phosphate. An X-ray investigation.

The X-ray structure analysis of three compounds of interest as enzyme substrates is reported. They are the hydrated forms of (I) DL-2-amino-4-arsonobutanoic acid [HO-AsO2--CH2-CH2-CH(NH3+)-CO2H], (II) DL-2-amino-4-phosphonobutanoic acid [HO-PO2--CH2-CH2-CH(NH3+)-CO2H] and the hydrated barium salt of (III) D-3-phosphoglycerate [HO-PO2--O-CH2-CH(OH)-CO2-]. The structures were fully refined to R factors of 0.033, 0.053 and 0.046. For the compounds (I) and (II) the charge distribution was directly determined by locating all H atoms. The co-ordination around As and P is approximately tetrahedral, with the valency angle between the two charged O atoms enlarged to 112 degrees in compound (I), 166 degrees in compound (II) and 122 degrees in compound (III). The As-X bond distances are increased relative to P-X to accommodate the increased atomic radius. The analysis establishes that the compounds are structural analogues. Tables of co-ordinates for H atoms, anisotropic thermal parameters, bond lengths and bond angles for the three compounds have been deposited as Supplementary Publication SUP 50122 (5 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained directly [see Biochem J. (1983) 209, 5].

Aminobutyrates↗
PubMedDate not supplied

Separation of phosphorylated sugars using capillary electrophoresis with indirect photometric detection.

Adenosine monophosphate (AMP) and naphthalene disulfonate (NDS) have been characterized as electrolytes for the indirect photometric detection of phosphorylated sugars and other organophosphorus compounds of biochemical interest. This work has resulted in the CE separation on an uncoated capillary using 5 mM AMP and 100 mM boric acid at pH 7.2 of six metabolites (glucose-6-phosphate [G6P], fructose-6-phosphate [F6P]), fructose-1,6-bisphosphate [F-1,6-P], dihydroxyacetone phosphate [DHAP], glyceraldehyde-3-phosphate [G3P], and 2-phosphoglycerate [2-PG] or 3-phosphoglycerate [3-PG]) found in the glycolytic pathway. The detection limits using a 5-sec injection time were between 0.5 and 1 mg/L for these compounds, with the exception of G3P. Resolution between 3-PG and 2-PG is possible by the addition of magnesium ion, although the separation time is longer. A successful separation of five monophosphorylated sugars (G6P, F6P, ribose-5-phosphate [R5P], sucrose-6-phosphate [S6P], and 2-PG) has been performed using the same conditions as for the glycolytic pathway separation. A separation of bisphosphorylated sugars (glucose-1,6-bisphosphate [G-1,6-P],F-1,6-P, ribulose-1,5-bisphosphate [Ru-1, 5P], and sedoheptulose-1,7-bisphosphate [S-1, 7P]) could not be performed with AMP unless magnesium chloride was added. With NDS, a separation of these bisphosphorylated sugars can be obtained without the addition of magnesium chloride.

Dihydroxyacetone Phosphate↗
PubMed2004

Both subunits of ADP-glucose pyrophosphorylase are regulatory.

The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits.

Adenosine Diphosphate Glucose↗
PubMed2003

Relative turnover numbers of maize endosperm and potato tuber ADP-glucose pyrophosphorylases in the absence and presence of 3-phosphoglyceric acid.

Adenosine diphosphate glucose pyrophosphorylase (AGPase; EC 2.7.7.27) synthesizes the starch precursor, ADP-glucose. It is a rate-limiting enzyme in starch biosynthesis and its activation by 3-phosphoglyceric acid (3PGA) and/or inhibition by inorganic phosphate (Pi) are believed to be physiologically important. Leaf, tuber and cereal embryo AGPases are highly sensitive to these effectors, whereas endosperm AGPases are much less responsive. Two hypotheses can explain the 3PGA activation differences. Compared to leaf AGPases, endosperm AGPases (i) lack the marked ability to be activated by 3PGA or (ii) they are less dependent on 3PGA for activity. The absence of purified preparations has heretofore negated answering this question. To resolve this issue, heterotetrameric maize ( Zea mays L.) endosperm and potato ( Solanum tuberosum L.) tuber AGPases expressed in Escherichia coli were isolated and the relative amounts of enzyme protein were measured by reaction to antibodies against a motif resident in both small subunits. Resulting reaction rates of both AGPases are comparable in the presence but not in the absence of 3PGA when expressed on an active-protein basis. We also placed the potato tuber UpReg1 mutation into the maize AGPase. This mutation greatly enhances 3PGA sensitivity of the potato AGPase but it has little effect on the maize AGPase. Thirdly, lysines known to bind 3PGA in potato tuber AGPase, but missing from the maize endosperm AGPase, were introduced into the maize enzyme. These had minimal effect on maize endosperm activity. In conclusion, the maize endosperm AGPase is not nearly as dependent on 3PGA for activity as is the potato tuber AGPase.

Enzyme Activation↗
PubMed2005

The regulation of Rubisco activity in response to variation in temperature and atmospheric CO2 partial pressure in sweet potato.

The temperature response of net CO(2) assimilation rate (A), the rate of whole-chain electron transport, the activity and activation state of Rubisco, and the pool sizes of ribulose-1,5-bisphosphate (RuBP) and 3-phosphoglyceric acid (PGA) were assessed in sweet potato (Ipomoea batatas) grown under greenhouse conditions. Above the thermal optimum of photosynthesis, the activation state of Rubisco declined with increasing temperature. Doubling CO(2) above 370 mubar further reduced the activation state, while reducing CO(2) by one-half increased it. At cool temperature (<16 degrees C), the activation state of Rubisco declined at CO(2) levels where photosynthesis was unaffected by a 90% reduction in O(2) content. Reduction of the partial pressure of CO(2) at cool temperature also enhanced the activation state of Rubisco. The rate of electron transport showed a pronounced temperature response with the same temperature optimum as A at elevated CO(2). RuBP pool size and the RuBP-to-PGA ratio declined with increasing temperature. Increasing CO(2) also reduced the RuBP pool size. These results are consistent with the hypothesis that the reduction in the activation state of Rubisco at high and low temperature is a regulated response to a limitation in one of the processes contributing to the rate of RuBP regeneration. To further evaluate this possibility, we used measured estimates of Rubisco capacity, electron transport capacity, and the inorganic phosphate regeneration capacity to model the response of A to temperature. At elevated CO(2), the activation state of Rubisco declined at high temperatures where electron transport capacity was predicted to be limiting, and at cooler temperatures where the inorganic phosphate regeneration capacity was limiting. At low CO(2), where Rubisco capacity was predicted to limit photosynthesis, full activation of Rubisco was observed at all measurement temperatures.

Carbon Dioxide↗
PubMed1996

2.0 A resolution structure of a ternary complex of pig muscle phosphoglycerate kinase containing 3-phospho-D-glycerate and the nucleotide Mn adenylylimidodiphosphate.

The crystal structure of a ternary complex of pig muscle phosphoglycerate kinase (PGK) containing 3-phosphoglycerate (3-PG) and manganese adenylylimidodiphosphate (Mn AMP-PNP) has been determined and refined at 2.0 A resolution. The complex differs from the true substrate ternary complex only in the presence of an imido- rather than an oxylink between beta- and gamma-phosphates of the bound nucleotide. The 3-PG is bound in a similar manner to that observed in binary complexes. The nucleotide is bound in a similar manner to Mg ADP except that the metal ion is coordinated by all three alpha-, beta-, and gamma-phosphates, but not by the protein. The gamma-phosphate, which is transferred in the reaction, is not bound by the protein. One further characteristic of the ternary complex is that Arg-38 moves to a position where its guanidinium group makes a triple interaction with the N-terminal domain, the C-terminal domain, and the 1-carboxyl group of the bound 3-PG. Although a hinge-bending conformation change is seen in the ternary complex, it is no larger than that observed in the 3-PG binary complex. To reduce that distance between two bound substrates to a value consistent with the direct in-line transfer known to occur in PGK, we modeled the closure of a pronounced cleft in the protein structure situated between the bound substrates. This closure suggested a mechanism of catalysis that involves the "capture" of the gamma-phosphate by Arg-38 and the N-terminus of helix-14, which has a conserved Gly-Gly-Gly phosphate binding motif. We propose that nucleophilic attack by the 1-carboxyl group of the 3-PG on the gamma-phosphorus follows the capture of the gamma-phosphate, leading to a pentacoordinate transition state that may be stabilized by hydrogen bonds donated by the NH groups in the N-terminus of helix 14 and the guanidinium group of Arg-38. During the course of the reaction the metal ion is proposed to migrate to a position coordinating the alpha- and beta-phosphates and the carboxyl group of Asp-374. The mechanism is consistent with the structural information from binary and ternary substrate complexes and much solution data, and gives a major catalytic role to Arg-38, as indicated by site-directed mutagenesis.

Adenylyl Imidodiphosphate↗
PubMed1975

The roots of bioenergetics.

Understanding metabolic energy transformation began with the realization of an 'intrusion' of phosphate into the mechanism of alcoholic fermentation. The discovery of an analogous participation of phosphate in muscle glycolysis connected the metabolic generation of energy-rich phosphate bonds fed into a common transmitter, adenosine triphosphate (ATP), with the production of mechanical energy through the finding that the phosphoryl group of creatine phosphate transferred to ATP could supply the energy for muscle contraction. In this way, a functional applicability of the energy of the phosphate bond was first shown. This observation was soon followed by the recognition that the phosphoanhydride bond of ATP provided the driving force in biosynthetic reactions; in this type of bond, metabolic energy apparently collects before it is transmitted for functional and biosynthetic use. The storage of energy in ATP was first detected in anaerobic energy-yielding reactions but soon was also found in respiratory and photosynthetic energy production. However, the mechanism by which energy derived from metabolites was converted into phosphate-bond energy in the latter processes appeared to differ from that of anaerobic energy transmission. Whereas phosphorylated compounds mediate the latter in homogeneous solutions, aerobic phosphorylation and photophosphorylation in prokaryotes seem to require special submembranous structures; and in eukaryotes, energy conversion is a function of special organelles, the mitochondria and chloroplasts. The evolutionary aspects of the transition from prokaryotes to eukaryotes are of considerable interest. In conclusion, the relevance of an apparent prokaryotic origin of the energy-transforming organelles in the eukaryotes will be commented on.

Adenosine Triphosphatases↗
PubMed1990

The effects of ligands on the conformation of phosphoglycerate kinase: fluorescence anisotropy decay and theoretical interpretation.

Horse muscle phosphoglycerate kinase (PGK) is a monomer folded into two widely distant domains. In the glycolytic pathway, this enzyme catalyzes the first reaction that produces ATP. It was suggested, by analogy with yeast hexokinase, that a hinge-bending motion may be induced by the binding of specific substrates to the protein. To analyze such a motion, or any structural changes induced by ligand binding, fluorescence anisotropy decay of tryptophan residues in free and liganded PGK was studied. At 293 K, for the free protein and the binary complex with 3-phosphoglycerate, a single correlation time of 26 ns was observed, corresponding to the rotation of the overall protein, whereas upon addition of MgADP, this correlation time decreased to 10 ns. Such a decrease cannot be merely due to a change of the protein's shape and volume. To explain this, it was suggested that the fluorescence anisotropy decay of the PGK-MgADP complex corresponded to the rotation of the only buried tryptophan (Trp 335). The rotational paths of this tryptophan, in the presence and absence of the nucleotide, were established by potential energy minimization calculations. The results indicated that MgADP induces a displacement of helix alpha-13 that decreases the rotational energy barrier of Trp 335 from 16 kcal/mol in the free protein to 8 kcal/mol in the complex.

Adenosine Diphosphate↗
PubMed2003

Protein stabilisation by compatible solutes: effect of mannosylglycerate on unfolding thermodynamics and activity of ribonuclease A.

Differential scanning calorimetry, optical spectroscopy, and activity measurements were used to investigate the effect of mannosylglycerate, a negatively charged osmolyte widely distributed among thermophilic and hyperthermophilic archaea and bacteria, on the thermal unfolding of ribonuclease A (RNase A). For comparison, assays in the presence of trehalose, a canonical solute in mesophiles, and potassium chloride were also carried out. A thermodynamic analysis was performed by using differential scanning calorimetry data. The changes in the heat capacity for unfolding were similar for the different solutes examined. Mannosylglycerate was an efficient thermostabiliser of RNase A and induced an increase of 6 degrees C mole(-1) in the melting temperature. Moreover, the performance of mannosylglycerate as a stabiliser depended on the net charge of the molecule, with the maximal effect being observed at pH values above 4.5. Analysis of the enthalpic and entropic contributions to unfolding, derived from calorimetric data, revealed that the stabilisation rendered by mannosylglycerate is primarily achieved through a decrease in the unfolding entropy. Also, the number of protons taken up by RNase A upon denaturation in the presence of mannosylglycerate was considerably higher than with other solutes, a result consistent with a more rigid structure of the native protein. Mannosylglycerate (potassium salt) inhibited the activity of RNase A, albeit to a smaller extent than KCl, and acted as an efficient suppressor of aggregation of the denatured protein, thereby having a remarkable beneficial effect on the inactivation of RNase A upon thermal denaturation. The results are discussed in view of the physiological role of this charged compatible solute.

Animals↗
PubMed1991

Conformational changes in yeast phosphoglycerate kinase upon ligand binding: fluorescence of a linked probe and chemical reactivity of genetically introduced cysteinyl residues.

The effects of ligands on the conformation of yeast phosphoglycerate kinase were explored by introducing cysteinyl residues at different positions in the molecule by site-directed mutagenesis. Thus several mutants were constructed, each containing a unique cysteinyl residue. Neither the conformation nor the enzyme activity was affected by the substitutions. The reactivity of the thiol groups and the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine covalently linked to these thiols were used to monitor the conformational changes induced upon ligand binding. It was found that the observed changes mainly involve the part of the protein located in the cleft, particularly the environment of residues 35 and 183. No alteration was observed on the external side of the protein. Only 3-Phosphoglycerate induced these conformational changes. However, when the fluorescent probe was attached to residue 377, the binding of the two substrates was required to induce a modification in the fluorescence of the probe. These results indicate that the substrates separately or together induce discrete molecular motions in phosphoglycerate kinase.

Adenosine Triphosphate↗
PubMed1993

Anion-exchange analysis of ribulose-bisphosphate carboxylase/oxygenase reactions: CO2/O2 specificity determination and identification of side products.

An improved anion-exchange chromatographic method for determining the carboxylation/oxygenation specificity (tau) of ribulose 1,5-bisphosphate carboxylase/oxygenase is presented. This assay, which entails radiometric detection of [1-3H]ribulose-bisphosphate turnover products separated on MonoQ anion-exchange resin, is more convenient, less error-prone, and more generally applicable than previous methods of tau determination. It is suitable for both wild-type and site-directed mutant enzymes of widely varying activity and specificity levels and allows simultaneous visualization of various side products of ribulose-bisphosphate processing. A facile method for scrubbing dissolved O2 from carboxylase reaction solutions, which does not require extensive purging and exchange of dissolved gases, is also described.

Anion Exchange Resins↗
PubMed1994

Time-course curves of enzyme reactions with dilute substrates can be determined from the reverse reactions.

We present a novel and useful principle that can be applied to enzyme reactions with dilute substrates and enables to obtain the desired time-course curve from the reverse reaction when the latter can be measured more easily. Let us imagine an enzyme mixture that can catalyze the interconversion between substances A, B, C,.... If one substance (sufficiently dilute) is added to the mixture as the substrate and the time-course curve of the concentration of another substance is followed, then, "the time-course curve of A-->B" (which is the time-course curve of the concentration of substance B when substance A is added as the substrate) and that of the reverse reaction B-->A will coincide when plotted at appropriate scales of magnitude. Similar relation holds on any pair of substances even if they are widely separated in any type of reaction scheme. The principle was proved mathematically and holds on any first-order reaction network and can be applied on an enzyme reaction with a dilute substrate because it is reduced to a first-order reaction network. An example of experimental application of this principle is also given.

Catalysis↗
PubMed1988

31P magnetization transfer in the phosphoglyceromutase-enolase coupled enzyme system.

The rate of exchange of phosphoryl groups between 2-phosphoglycerate, 3-phosphoglycerate and phosphoenolpyruvate by the coupled phosphoglyceromutase-enolase enzyme system using one- and two-dimensional 31P NMR spectroscopy was measured. Magnetization exchange in one-dimensional experiments was achieved by saturation transfer with selective irradiation at both one and two sites in this three-site exchange system using the DANTE pulse sequence. The two-dimensional magnetization exchange experiment avoids the need to selectively saturate at one or more frequencies which may be difficult in complex exchange systems. Analysis of the two-dimensional exchange experiment by the back transformation method yielded exchange rate constants in good agreement with the saturation transfer method.

Animals↗