[Malate dehydrogenase, lactate dehydrogenase, pyruvate kinase, phosphoglycerate kinase and aspartate aminotransferase in the dentin of cattle of various ages. Preliminary note].
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Animals↗
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Phosphoglycerate kinase.
Phosphoglycerate kinase catalyses the high-energy phosphoryl transfer of the acyl phosphate of 1,3-bisphosphoglycerate to ADP to produce ATP, a reaction requiring magnesium ions. The enzyme is widely distributed and apparently highly conserved as a monomer of molecular mass 45 000. X-ray studies of the enzymes from horse muscle and yeast, carried out in Oxford and Bristol respectively, have shown that the molecular structures of the two enzymes are almost identical. The most striking aspect of the structure is that the single polypeptide chain is organized into two separated domains composed of the N-terminal and C-terminal halves of the chain. Substrate binding studies and the determination of the complete amino acid sequence of the horse enzyme suggest that the nucleotide substrates and the phosphoglycerate substrates are bound to the C-domain and N-domain, respectively, in sites that are separated by about 12 A. In order to bring the two substrates together for catalysis, a hinge-bending conformational change involving helix rotation has been proposed, for which there is independent evidence from solution studies. Crystals of the ternary complex of the horse enzyme have been prepared that may contain the folded form of the enzyme.
Amino Acid Sequence↗
Calmodulin binds to and inhibits the activity of phosphoglycerate kinase.
Phosphoglycerate kinase (PGK) functions as a cytoplasmic ATP-generating glycolytic enzyme, a nuclear mediator in DNA replication and repair, a stimulator of Sendai virus transcription and an extracellular disulfide reductase in angiogenesis. Probing of a developmental expression library from Dictyostelium discoideum with radiolabelled calmodulin led to the isolation of a cDNA encoding a putative calmodulin-binding protein (DdPGK) with 68% sequence similarity to human PGK. Dictyostelium, rabbit and yeast PGKs bound to calmodulin-agarose in a calcium-dependent manner while DdPGK constructs lacking the calmodulin-binding domain (209KPFLAILGGAKVSDKIKLIE228) failed to bind. The calmodulin-binding domain shows 80% identity between diverse organisms and is situated beside the hinge and within the ATP binding domain adjacent to nine mutations associated with PGK deficiency. Calmodulin addition inhibits yeast PGK activity in vitro while the calmodulin antagonist W-7 abrogates this inhibition. Together, these data suggest that PGK activity may be negatively regulated by calcium and calmodulin signalling in eukaryotic cells.
Amino Acid Sequence↗
Isolation and DNA sequence of a full-length cDNA clone for human X chromosome-encoded phosphoglycerate kinase.
Phosphoglycerate kinase (PGK), a major enzyme in glycolysis, is encoded by the X chromosome in mammals. We have initiated molecular analysis of the PGK structural gene by isolating a full-length cDNA clone from a human fetal liver cDNA library. Synthetic oligonucleotide mixtures encoding two different portions of PGK were used as direct in situ hybridization probes for the cDNA library. Several classes of clones were obtained based on their hybridization at different stringencies to one or both of the PGK oligonucleotide mixtures. One clone, designated pHPGK-7e, which hybridized at high stringency to each of the synthetic probes, encoded the complete PGK protein sequence as well as 82 base pairs of 5' and 437 base pairs of 3' untranslated regions. Southern blot analysis of human genomic DNAs revealed a complex pattern of hybridizing fragments, two of which were non-X in origin. These results suggest that the human genome contains a small family of dispersed PGK or PGK-like genes.
Amino Acid Sequence↗
Effects of C-terminal deletions on the conformational state and denaturation of phosphoglycerate kinase.
Phosphoglycerate kinase (PGK) contains two domains of approximately equal size, both of the alpha/beta type. An alpha-helix consisting of the middle section of the 415-amino acid polypeptide chain, and the N- and C-termini reside in the interdomain hinge region [Watson, H. C., et al. (1982) EMBO J. 1, 1635-1640]. The C-terminal end is an integral part of the N-terminal domain. The consequences of the deletion of fifteen and three C-terminal amino acids on the conformational state and on the guanidine hydrochloride-induced and thermal unfolding of PGK were investigated by using near- and far-UV CD, tryptophan fluorescence, 1-anilinonaphthalene-8-sulfonic acid binding, accessibility to chemical modification, and differential scanning calorimetry. The results of these studies indicate that the conformations of both domains and of the interdomain region were altered by these deletions. In the absence of the 15-amino acid C-terminal peptide [delta(401-415)], the N-terminal domain exhibits several characteristics of a molten globule state, whereas the C-terminal domain retains native-like, although distinctly different, tertiary structure. Deletion of three C-terminal amino acids [delta(413-415)] also globally affects PGK conformation, although to a much lesser extent. Both C-terminal deletions resulted in a significant decrease in protein stability, as demonstrated by their increased susceptibility to guanidine-induced and thermal denaturation. These results suggest that the formation of a native tertiary fold of PGK requires the presence of a complete polypeptide chain.
Calorimetry, Differential Scanning↗
Adenine nucleotides and some related enzyme activities (adenylate kinase and phosphoglycerate kinase) in normal and abnormal human semen.
Adenine nucleotides (ATP and ADP) and enzyme activities of Adenylate Kinase (AK) and Phosphoglycerate Kinase (PGK) were assayed on semen samples from normal and infertile men. In accordance with the values obtained, samples could be classified into two groups: one group with levels of either adenine nucleotides and enzymes assumed as normal; a second group with high levels of all the parameters. The latter group comprised the samples with low sperm density (less than 40 million/ml). When the samples were subdivided according to sperm motility, the above distinction into two groups appeared less evident. Moreover, comparing biochemical parameters with morphological findings some contrasts were seen. Nevertheless, we suggest comparing both biochemical and morphological data for a more detailed classification of human semen abnormalities.
Adenine Nucleotides↗
Simultaneous purification of glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and phosphoglycerate mutase from pig liver and muscle.
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Animals↗
Dimeric 3-phosphoglycerate kinases from hyperthermophilic Archaea. Cloning, sequencing and expression of the 3-phosphoglycerate kinase gene of Pyrococcus woesei in Escherichia coli and characterization of the protein. Structural and functional comparison with the 3-phosphoglycerate kinase of Methanothermus fervidus.
The gene coding for the 3-phosphoglycerate kinase (EC 2.7.2.3) of Pyrococcus woesei was cloned and sequenced. The gene sequence comprises 1230 bp coding for a polypeptide with the theoretical M(r) of 46,195. The deduced protein sequence exhibits a high similarity (46.1% and 46.6% identity) to the other known archaeal 3-phosphoglycerate kinases of Methanobacterium bryantii and Methanothermus fervidus [Fabry, S., Heppner, P., Dietmaier, W. & Hensel, R. (1990) Gene 91, 19-25]. By comparing the 3-phosphoglycerate kinase sequences of the mesophilic and the two thermophilic Archaea, trends in thermoadaptation were confirmed that could be deduced from comparisons of glyceraldehyde-3-phosphate dehydrogenase sequences from the same organisms [Zwickl, P., Fabry, S., Bogedain, C., Haas, A. & Hensel, R. (1990) J. Bacteriol. 172, 4329-4338]. With increasing temperature the average hydrophobicity and the portion of aromatic residues increases, whereas the chain flexibility as well as the content in chemically labile residues (Asn, Cys) decreases. To study the phenotypic properties of the 3-phosphoglycerate kinases from thermophilic Archaea in more detail, the 3-phosphoglycerate kinase genes from P. woesei and M. fervidus were expressed in Escherichia coli. Comparisons of kinetic and molecular properties of the enzymes from the original organisms and from E. coli indicate that the proteins expressed in the mesophilic host are folded correctly. Besides their higher thermostability according to their origin from hyperthermophilic organisms, both enzymes differ from their bacterial and eucaryotic homologues mainly in two respects. (a) The 3-phosphoglycerate kinases from P. woesei and M. fervidus are homomeric dimers in their native state contrary to all other known 3-phosphoglycerate kinases, which are monomers including the enzyme from the mesophilic Archaeum M. bryantii. (b) Monovalent cations are essential for the activity of both archaeal enzymes with K+ being significantly more efficient than Na+. For the P. woesei enzyme, non-cooperative K+ binding with an apparent Kd (K+) of 88 mM could be determined by kinetic analysis, whereas for the M. fervidus 3-phosphoglycerate kinase the K+ binding is rather complex: from the fitting of the saturation data, non-cooperative binding sites with low selectivity for K+ and Na+ (apparent Kd = 270 mM) and at least three cooperative and highly specific K+ binding sites/subunit are deduced. At the optimum growth temperature of P. woesei (100 degrees C) and M. fervidus (83 degrees C), the 3-phosphoglycerate kinases show half-lives of inactivation of only 28 min and 44 min, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Quantitative histochemistry of the primate skin. IX. Phosphoglycerate kinase and phosphoglycerate mutase.
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Animals↗
[Simultaneous purification of D-glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and phosphoglycerate phosphomutase from Escherichia coli].
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Escherichia coli↗
Tissue levels of glycolytic enzymes in phosphoglycerate kinase deficiency.
Phosphoglycerate kinase deficiency is a rate, X-linked disorder associated with a severe haemolytic anaemia. In general the deficiency has been demonstrated only in erythrocytes and leucocytes. However, in a subject with this condition, the activity of phosphoglycerate kinase in lymphocytes and platelets was also shown to be less than 5% of the normal value. Following the death of this subject in 1979, the deficiency was also found to occur in tissue samples of brain, skeletal muscle, liver and cardiac muscle, obtained at the autopsy. Values for phosphoglycerate kinase were of the order of 0.5-5% of normal controls. Other glycolytic enzymes which were tested were hexokinase, pyruvate kinase, enolase and 2-phosphoglyceromutase. In general, values for these enzymes were either normal or slightly raised.
Adolescent↗
Structural and functional properties of mutant Arg203Pro from yeast phosphoglycerate kinase, as a model of phosphoglycerate kinase-Uppsala.
A pathological variant of human phosphoglycerate kinase, phosphoglycerate kinase-Uppsala, associated with chronic nonspherocytic hemolytic anemia has been found to differ from the normal enzyme by substitution of an arginine at position 206 (corresponding to position 203 in yeast) by a proline. In order to understand the structural and functional consequences of this mutation, the corresponding mutant in yeast phosphoglycerate kinase was constructed. The three-dimensional structure of this mutant was resolved at 2.9 A. Although the overall structure is not modified, small local changes were observed. The kinetic parameters of the mutant were not found to be greatly affected, the catalytic constant being lowered by only 10-20%. The most significant difference when compared with the wild-type enzyme is a decrease in stability by about 3 kcal/mol. The physiological implications of this instability are discussed.
Circular Dichroism↗
Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation.
Phosphoglycerate kinase (PGK), a key enzyme in glycolysis, catalyses the transfer of a phosphoryl-group from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP. Despite extensive kinetic and structural investigations over more than two decades, the conformation assumed by this enzyme during catalysis remained unknown. Here we present the 2.8 A crystal structure of a ternary complex of PGK from Trypanosoma brucei, the causative agent of sleeping sickness. This structure determination relied on a procedure in which fragments containing less than 10% of the scattering mass were successively positioned in the unit cell to obtain phases. The PGK ternary complex exhibits a dramatic closing of the large cleft between the two domains seen in all previous studies, thereby bringing the two ligands, 3-phosphoglycerate and ADP into close proximity. Our results demonstrate that PGK is a hinge-bending enzyme, reveal a novel mechanism in which substrate-induced effects combine synergistically to induce major conformational changes and, to our knowledge, afford the first observation of the PGK active site in a catalytic conformation.
Adenosine Diphosphate↗
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.
Adenosine Triphosphate↗
Correlation between conformational stability of the ternary enzyme-substrate complex and domain closure of 3-phosphoglycerate kinase.
3-phosphoglycerate kinase (PGK) is a typical two-domain hinge-bending enzyme with a well-structured interdomain region. The mechanism of domain-domain interaction and its regulation by substrate binding is not yet fully understood. Here the existence of strong cooperativity between the two domains was demonstrated by following heat transitions of pig muscle and yeast PGKs using differential scanning microcalorimetry and fluorimetry. Two mutants of yeast PGK containing a single tryptophan fluorophore either in the N- or in the C-terminal domain were also studied. The coincidence of the calorimetric and fluorimetric heat transitions in all cases indicated simultaneous, highly cooperative unfolding of the two domains. This cooperativity is preserved in the presence of substrates: 3-phosphoglycerate bound to the N domain or the nucleotide (MgADP, MgATP) bound to the C domain increased the structural stability of the whole molecule. A structural explanation of domain-domain interaction is suggested by analysis of the atomic contacts in 12 different PGK crystal structures. Well-defined backbone and side-chain H bonds, and hydrophobic and electrostatic interactions between side chains of conserved residues are proposed to be responsible for domain-domain communication. Upon binding of each substrate newly formed molecular contacts are identified that firstly explain the order of the increased heat stability in the various binary complexes, and secondly describe the possible route of transmission of the substrate-induced conformational effects from one domain to the other. The largest stability is characteristic of the native ternary complex and is abolished in the case of a chemically modified inactive form of PGK, the domain closure of which was previously shown to be prevented [Sinev MA, Razgulyaev OI, Vas M, Timchenko AA & Ptitsyn OB (1989) Eur J Biochem180, 61-66]. Thus, conformational stability correlates with domain closure that requires simultaneous binding of both substrates.
Adenosine Diphosphate↗
Regenerating liver in aged rats produces unaltered phosphoglycerate kinase.
Pure phosphoglycerate kinase from old rat liver shows altered properties. However, when old animals are partially hepatectomized, the regenerating liver produces the "young" form of the enzyme. By 4 or 5 days after hepatectomy, the enzyme once again becomes "old" in its properties. The results are interpreted as showing that normal enzyme is produced that becomes post-synthetically modified. This interpretation agrees with the idea that in old animals, "young" enzymes become conformationally modified, perhaps because of a slowing of protein turnover.
Aging↗
Magnetic isotope effect of magnesium in phosphoglycerate kinase phosphorylation.
Phosphoglycerate kinase (PGK) is found to be controlled by a (25)Mg(2+)-related magnetic isotope effect. Mg(2+) nuclear spin selectivity manifests itself in PGK-directed ADP phosphorylation, which has been clearly proven by comparison of ATP synthesis rates estimated in reaction mixtures with different Mg isotopy parameters. Both pure (25)Mg(2+) (nuclear spin 5/2, magnetic moment +0.85) and (24)Mg(2+) (spinless, nonmagnetic nucleus) species as well as their mixtures were used in experiments. In the presence of (25)Mg(2+), ATP production is 2.6 times higher compared with the yield of ATP reached in (24)Mg(2+)-containing PGK-based catalytic systems. The chemical mechanism of this phenomenon is discussed. A key element of the mechanism proposed is a nonradical pair formation in which (25)Mg(+) radical cation and phosphate oxyradical are involved.
Adenosine Diphosphate↗
Role of loops in the folding and stability of yeast phosphoglycerate kinase.
Yeast phosphoglycerate kinase (yPGK) is a monomeric two domain protein used as folding model representative of large proteins. We inserted short unstructured sequences (four Gly or four Thr) into the connections between secondary structure elements and studied the consequences of these insertions on the folding process and stability of yPGK. All the mutated proteins can refold efficiently. The effect per residue on stability is larger for the first inserted residue. Insertion in two long betaalpha loops (at residue positions 71 and 129) is more destabilizing than an insertion in a short alphabeta loop (at residue position 89) located on the opposite side of the N-terminal domain. The effect on stability is mainly due to a large increase of the unfolding rate rather than a decrease of the folding rate. This suggests that these connections between secondary structure elements do not play an active role in directing the folding process. Insertion into the short alphabeta loop (position 89) has limited effects on stability and results in the detection of a kinetic phase not previously seen with the wild-type protein, suggesting that insertions in this particular loop do qualitatively affect the folding process without a large effect on folding efficiency. For the two long betaalpha loops (positions 71 and 129) located in the inner surface of the N-terminal domain, the effects on stability are possibly associated with decoupling of the two domains as observed by differential scanning calorimetry during thermal unfolding.
Enzyme Stability↗