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PubMed1979

Spectrophotometric studies on the interaction between triose phosphate isomerase and inhibitors.

The binding of ligands to chicken muscle triose phosphate isomerase was studied. Changes in u.v. absorbance of the enzyme were used to measure binding, and the dissociation constant was determined over a range of pH values. The ligands were 2-phosphoglycollate and rac-glycerol 3-phosphate (only the D-isomer, sn-glycerol 1-phosphate, binds appreciably). Non-linear regression was used to fit calculated curves to the experimental points and hence to compare different models. Both active sites in the dimeric enzyme probably bound 2-phosphoglycollate, without any interaction between the sites. The results of crystallographic analysis [phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc Trans. 5, 642--647], and experiments on the 1H, 13C and 31P n.m.r. of enzyme or 2-phosphoglycollate were combined with the present results to provide the basis for a model in which binding depends on glutamic acid-165 being protonated and on the ligant being fully ionized; additionally, binding affects the ionization of one histidine residue (probably histidine-100). The binding of the glycerol 3-phosphate, on the other hand, was independent of pH over the range pH 6.5--8.5 but decreased at lower pH values. This is explained on a model in which the binding of the monoanion of the ligand is markedly affected by the protonation of a residue in the enzyme, but the binding of the dianion is only slightly affected by this ionization.

Animals↗
PubMed2003

Triosidines: novel Maillard reaction products and cross-links from the reaction of triose sugars with lysine and arginine residues.

The role of the highly reactive triose sugars glyceraldehyde and glyceraldehyde-3-phosphate in protein cross-linking and other amino acid modifications during the Maillard reaction was investigated. From the incubation of glyceraldehyde with N (alpha)-acetyl-L-lysine and N (alpha)-acetyl-L-arginine, we isolated four new Maillard reaction pyridinium compounds named 'triosidines'. Two of them, 'lys-hydroxy-triosidine' [1-(5-amino-5-carboxypentyl)-3-[(5-amino-5-carboxypentylamino)methyl]-5-hydroxypyridinium] and 'arg-hydroxy-triosidine' [2-(4-amino-4-carboxybutylamino)-8-(5-amino-5-carboxypentyl)-6-hydroxy-3,4-dihydro-pyrido[2,3-d]pyrimidin-8-ium] are fluorescent, UV-active Lys-Lys and Lys-Arg cross-links respectively. Their structures were identified by NMR and MS. In addition, two UV-active lysine adducts, 'trihydroxy-triosidine' [1-(5-amino-5-carboxypentyl)-3,4-dihydroxy-5-(hydroxymethyl)pyridinium] and 'triosidine carbaldehyde' [1-(5-amino-5-carboxypentyl)-3-formylpyridinium] were tentatively identified by MS. All structures involve six sugar-derived carbons as part of the heterocyclic ring. Of the two novel cross-links, only arg-hydroxy-triosidine was formed by glyceraldehyde-3-phosphate, an intermediate metabolite of the glycolytic pathway. Lys-hydroxy-triosidine and arg-hydroxy-triosidine were detected in human and porcine corneas treated with glyceraldehyde. The HPLC-fluorescence identification was confirmed by MS. Triosidines were also formed from dihydroxyacetone, a widely used artificial sun-tanning agent. Triosidines are expected to be useful tools in tissue engineering, where the utilization of highly reactive sugars is needed to stabilize the loose matrix. In addition, they are expected to be present in selected biological conditions, such as on consumption of a high fructose diet, and syndromes associated with high glyceraldehyde excretion, such as Fanconi Syndrome, fructose-1,6-diphosphatase deficiency and tyrosinaemia.

Amino Acids↗
PubMed1986

Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally altered protein in which a glutamate residue is replaced by an aspartate residue. The importance of glutamate-104 to enzyme structure and function is implicated by its conservation in the TPI protein of all species that have been characterized to date. The glutamate-to-aspartate substitution results in a thermolabile enzyme as demonstrated by assays of TPI activity in cultured fibroblasts of each patient and cultured Chinese hamster ovary (CHO) cells that were stably transformed with the mutant alleles. Although this substitution conserves the overall charge of amino acid-104, the x-ray crystal structure of chicken TPI indicates that the loss of a side-chain methylene group (-CH2CH2COO- ---- -CH2COO-) is sufficient to disrupt the counterbalancing of charges that normally exists within a hydrophobic pocket of the native enzyme.

Alleles↗
PubMed1992

cDNA cloning and functional expression of the Schistosoma mansoni protective antigen triose-phosphate isomerase.

M.1 monoclonal antibody has previously been shown to passively transfer partial resistance to schistosome infection within mice and to recognize a 28-kDa antigen that has peptide sequence homology with triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1). We have now isolated the complete coding DNA for Schistosoma mansoni TPI and confirmed that this cDNA encodes the 28-kDa antigen recognized by M.1. The predicted translation product has strong homology with other TPIs, particularly from higher eukaryotes, and the sequence homology is greatest in regions known to form the active site. The complete coding DNA has been expressed within an Escherichia coli host to produce high levels of soluble, recombinant S. mansoni TPI protein. The product is recognized and purified by the M.1 antibody and is a functional TPI with an intrinsic specific activity comparable to that of rabbit and yeast TPI.

Amino Acid Sequence↗
PubMed1995

Erythrocyte lipids in triose-phosphate isomerase deficiency.

Marked hypoalphalipoproteinemia was found together with relatively low serum cholesterol, triacylglycerol, and LDL levels in a triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1)-deficient Hungarian family, especially in the two compound-heterozygote brothers. Apart from a slight increase in palmitic and stearic acids together with a slight decrease in oleic and linoleic acids, no other changes were found in the fatty acid composition of the erythrocyte phospholipids. Anisotropy measurements with n-(9-anthroyloxy) stearic and -palmitic acid fluorophores revealed increased motional freedom of the fatty acid chains in the external lipid layers of the intact erythrocytes from all members of the TPI-deficient family as compared with normal age-matched controls. This asymmetric increase in membrane fluidity was found to be significantly higher in the propositus than in his compound-heterozygote brother without any neurological disorders. The change in membrane fluidity may result from as-yet-unresolved aspects of the lipid composition of the plasma membrane. Our findings that the differences between the TPI-deficient individuals and normal controls and the differences between the two compound-heterozygote brothers were all absent in the phospholipid extracts of the same erythrocytes favor the assumption that the increased motional freedom of the fatty acid chains in the external surface of the bilayer is caused by the binding of the mutant TPI molecule to the N-terminal sequence of band 3 protein.

Adult↗
PubMed1995

Evidence against the exon theory of genes derived from the triose-phosphate isomerase gene.

The exon theory of genes proposes that the introns of protein-encoding nuclear genes are remnants of the DNA spacers between ancient minigenes. The discovery of an intron at a predicted position in the triose-phosphate isomerase (EC 5.3.1.1) gene of Culex mosquitoes has been hailed as an evidential pillar of the theory. We have found that that intron is also present in Aedes mosquitoes, which are closely related to Culex, but not in the phylogenetically more distant Anopheles, nor in the fly Calliphora vicina, nor in the moth Spodoptera littoralis. The presence of this intron in Culex and Aedes is parsimoniously explained as the result of an insertion in a recent common ancestor of these two species rather than as the remnant of an ancient intron. The absence of the intron in 19 species of very diverse organisms requires at least 10 independent evolutionary losses in order to be consistent with the exon theory.

Amino Acid Sequence↗
PubMed1995

Seven newly discovered intron positions in the triose-phosphate isomerase gene: evidence for the introns-late theory.

The gene encoding the glycolytic enzyme triose-phosphate isomerase (TPI; EC 5.3.1.1) has been central to the long-standing controversy on the origin and evolutionary significance of spliceosomal introns by virtue of its pivotal support for the introns-early view, or exon theory of genes. Putative correlations between intron positions and TPI protein structure have led to the conjecture that the gene was assembled by exon shuffling, and five TPI intron positions are old by the criterion of being conserved between animals and plants. We have sequenced TPI genes from three diverse eukaryotes--the basidiomycete Coprinus cinereus, the nematode Caenorhabditis elegans, and the insect Heliothis virescens--and have found introns at seven novel positions that disrupt previously recognized gene/protein structure correlations. The set of 21 TPI introns now known is consistent with a random model of intron insertion. Twelve of the 21 TPI introns appear to be of recent origin since each is present in but a single examined species. These results, together with their implication that as more TPI genes are sequenced more intron positions will be found, render TPI untenable as a paradigm for the introns-early theory and, instead, support the introns-late view that spliceosomal introns have been inserted into preexisting genes during eukaryotic evolution.

Amino Acid Sequence↗
PubMed1998

Triose-phosphate isomerase (TIM) of the psychrophilic bacterium Vibrio marinus. Kinetic and structural properties.

The purification and characterization of triose-phosphate isomerase from the psychrophilic bacterium Vibrio marinus (vTIM) is described. Crystal structures of the vTIM-sulfate complex and the vTIM-2-phosphoglycolate complex (at a 2.7-A resolution) are also presented. The optimal growth temperature of Vibrio marinus is 15 degrees C. Stability studies show that vTIM is an unstable protein with a half-life of only 10 min at 25 degrees C. The vTIM sequence is most closely related to the sequence of Escherichia coli TIM (eTIM) (66% identity), and several unique structural features described for eTIM are also seen in vTIM, but eTIM is considerably more stable. The Td values of vTIM and eTIM, determined by calorimetric studies, are 41 and 54 degrees C, respectively. Amino acid sequence comparison reveals that vTIM has an alanine in loop 8 (at position 238), whereas all other TIM sequences known to date have a serine. The vTIM mutant A238S was produced and characterized. Compared with wild type, the catalytic efficiency of the A238S mutant is somewhat reduced, and its stability is considerably increased.

Amino Acid Sequence↗
PubMed1999

Lys13 plays a crucial role in the functional adaptation of the thermophilic triose-phosphate isomerase from Bacillus stearothermophilus to high temperatures.

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair normally present in mesophilic TIMs. His12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys13 is unknown. To investigate the role of the His12 and Lys13 pair in the enzyme's thermoadaptation, we reintroduced the "mesophilic residues" Asn and Gly into both thermophilic TIMs. Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His12 and Lys13 cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-A resolution. A molecular dynamics simulation showed that upon substitution of Lys13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys10. This suggests that Lys13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption.

Adaptation, Physiological↗
PubMed2002

Inhibition of plasmodium falciparum triose-phosphate isomerase by chemical modification of an interface cysteine. Electrospray ionization mass spectrometric analysis of differential cysteine reactivities.

Plasmodium falciparum triose-phosphate isomerase, a homodimeric enzyme, contains four cysteine residues at positions 13, 126, 196, and 217 per subunit. Among these, Cys-13 is present at the dimer interface and is replaced by methionine in the corresponding human enzyme. We have investigated the effect of sulfhydryl labeling on the parasite enzyme, with a view toward developing selective covalent inhibitors by targeting the interface cysteine residue. Differential labeling of the cysteine residues by iodoacetic acid and iodoacetamide has been followed by electrospray ionization mass spectrometry and positions of the labels determined by analysis of tryptic fragments. The rates of labeling follows the order Cys-196 > Cys-13 Cys-217/Cys-126, which correlates well with surface accessibility calculations based on the enzyme crystal structure. Iodoacetic acid labeling leads to a soluble, largely inactive enzyme, whereas IAM labeling leads to precipitation. Carboxyl methylation of Cys-13 results in formation of monomeric species detectable by gel filtration. Studies with an engineered C13D mutant permitted elucidation of the effects of introducing a negative charge at the interface. The C13D mutant exhibits a reduced stability to denaturants and 7-fold reduction in the enzymatic activity even under the concentrations in which dimeric species are observed.

Amino Acid Sequence↗
PubMed2005

The glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, and pyruvate kinase are components of the K(ATP) channel macromolecular complex and regulate its function.

The regulation of ATP-sensitive potassium (K(ATP)) channel activity is complex and a multitude of factors determine their open probability. Physiologically and pathophysiologically, the most important of these are intracellular nucleotides, with a long-recognized role for glycolytically derived ATP in regulating channel activity. To identify novel regulatory subunits of the K(ATP) channel complex, we performed a two-hybrid protein-protein interaction screen, using as bait the mouse Kir6.2 C terminus. Screening a rat heart cDNA library, we identified two potential interacting proteins to be the glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triose-phosphate isomerase. The veracity of interaction was verified by co-immunoprecipitation techniques in transfected mammalian cells. We additionally demonstrated that pyruvate kinase also interacts with Kir6.2 subunits. The physiological relevance of these interactions is illustrated by the demonstration that native Kir6.2 protein similarly interact with GAPDH and pyruvate kinase in rat heart membrane fractions and that Kir6.2 protein co-localize with these glycolytic enzymes in rat ventricular myocytes. The functional relevance of our findings is demonstrated by the ability of GAPDH or pyruvate kinase substrates to directly block the K(ATP) channel under patch clamp recording conditions. Taken together, our data provide direct evidence for the concept that key enzymes involved in glycolytic ATP production are part of a multisubunit K(ATP) channel protein complex. Our data are consistent with the concept that the activity of these enzymes (possibly by ATP formation in the immediate intracellular microenvironment of this macromolecular K(ATP) channel complex) causes channel closure.

Adenosine Triphosphate↗
PubMedDate not supplied

Inheritance and subcellular localization of triose-phosphate isomerase in dwarf mountain pine (Pinus mugo).

Several trees with expected heterozygous phenotype for triose-phosphate isomerase (TPI) were discovered in a population of dwarf mountain pine (Pinus mugo Turra) from southern Poland. As the inheritance of this enzyme in pines has not been reported, segregation of allelic variants was tested in eight trees with putative heterozygous phenotypes for two loci, TpiA and TPIB: Linkage between these and some other isozyme loci were studied and evidence for linkage has been found between TpiA and PgdA (r = 0.10) and between TpiB and DiaD (r = 0.36), but in single trees only. The subcellular localization of TPI isozymes was determined by comparing isoenzymes from the total extract with those found in fraction enriched in plastids, prepared by differential gradient centrifugation of cellular organelles. The more slowly migrating TPI-B isozyme is located in plastids.

Chromosome Segregation↗
PubMed2003

The sugar-metabolic enzymes aldolase and triose-phosphate isomerase are targets of glutathionylation in Arabidopsis thaliana: detection using biotinylated glutathione.

GSH has multiple actions in physiological responses of plants, but the molecular mechanisms are not fully understood. GSH plays an important role in functional alteration of proteins by reversible covalent incorporation (glutathionylation) in vertebrate cells. To investigate the function of glutathionylation in plant cells, we examined glutathionylated proteins in the suspension-cultured cells of Arabidopsis using biotinylated GSH. Biotinylated GSH was incorporated into about 20 proteins. Two of these proteins were identified as the key enzymes for sugar metabolism, triose-phosphate isomerase (TPI) and putative plastidic aldolase. Recombinant TPI was inactivated by GSSG, and it was reactivated by GSH. The physiological roles of glutathionylation of TPI and aldolase in sugar metabolism are discussed.

Aldehyde-Lyases↗
PubMed1981

On the three-dimensional structure and catalytic mechanism of triose phosphate isomerase.

Triose phosphate isomerase is a dimeric enzyme of molecular mass 56 000 which catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate. The crystal structure of the enzyme from chicken muscle has been determined at a resolution of 2.5 A, and an independent determination of the structure of the yeast enzyme has just been completed at 3 A resolution. The conformation of the polypeptide chain is essentially identical in the two structures, and consists of an inner cylinder of eight strands of parallel beta-pleated sheet, with mostly helical segments connecting each strand. The active site is a pocket containing glutamic acid 165, which is believed to act as a base in the reaction. Crystallographic studies of the binding of DHAP to both the chicken and the yeast enzymes reveal a common mode of binding and suggest a mechanisms for catalysis involving polarization of the substrate carbonyl group.

Animals↗
PubMed2000

Preliminary X-ray diffraction studies of rabbit muscle triose phosphate isomerase (TIM).

Triose phosphate isomerase (TIM) is responsible for the interconversion between GAP and DHAP in the glycolytic pathway. Two crystal forms belonging to space group P2(1)2(1)2(1) were obtained by the hanging-drop method and were designated A and B. Synchrotron X-ray diffraction data were collected for both forms. Form A had unit-cell parameters a = 65.14, b = 72.45, c = 93.24 A and diffracted to 2.25 A at 85 K, whereas form B had unit-cell parameters a = 73.02, b = 79.80, c = 172.85 A and diffracted to 2.85 A at room temperature. Molecular replacement was employed to solve the structures, using human TIM as a search model. Further refinement of both structures is under way and is expected to shed light on the recently reported conformational studies for rabbit TIM.

Animals↗
PubMed1986

Triose phosphate isomerase deficiency: report of a family.

Triose phosphate isomerase (TPI) deficiency is associated with a syndrome of congenital non-spherocytic haemolytic anaemia, mental subnormality, motor impairment, growth failure and cardiac failure. The deficiency state is characterized by moderately reduced red cell TPI activity, and marked instability of the abnormal enzyme to heat. The clinical features of an affected child are described, and some problems in the laboratory diagnosis delineated.

Carbohydrate Epimerases↗
PubMed2006

Triose phosphate isomerase, a novel enzyme-crystallin, and tau-crystallin in crocodile cornea. High accumulation of both proteins during late embryonic development.

Several enzymes are known to accumulate in the cornea in unusually high concentrations. Based on the analogy with lens crystallins, these enzymes are called corneal crystallins, which are diverse and species-specific. Examining crystallins in lens and cornea in multiple species provides great insight into their evolution. We report data on major proteins present in the crocodile cornea, an evolutionarily distant taxon. We demonstrate that tau-crystallin/alpha-enolase and triose phosphate isomerase (TIM) are among the major proteins expressed in the crocodile cornea as resolved by 2D gel electrophoresis and identified by MALDI-TOF. These proteins might be classified as putative corneal crystallins. tau-Crystallin, known to be present in turtle and crocodile lens, has earlier been identified in chicken and bovine cornea, whereas TIM has not been identified in the cornea of any species. Immunostaining showed that tau-crystallin and TIM are concentrated largely in the corneal epithelium. Using western blot, immunofluorescence and enzymatic activity, we demonstrate that high accumulation of tau-crystallin and TIM starts in the late embryonic development (after the 24th stage of embryonic development) with maximum expression in a two-week posthatched animal. The crocodile corneal extract exhibits significant alpha-enolase and TIM activities, which increases in the corneal extract with development. Our results establishing the presence of tau-crystallin in crocodile, in conjunction with similar reports for other species, suggest that it is a widely prevalent corneal crystallin. Identification of TIM in the crocodile cornea reported here adds to the growing list of corneal crystallins.

Alligators and Crocodiles↗