Genetic control of triosephosphate isomerase isoenzymes in wheat and rye

Summary The patterns of chloroplastic and cytosolic isoenzymes of triosephosphate isomerase were analysed by immunoblotting in leaves of rye, wheat, and some species of Aegilops or Agropyrum. While rye contained solely one chloroplastic and one cytosolic isoenzyme, wheat had a much more complex pattern which can be explained by the presence of three genomes in 6 x wheats (AABBDD) with distinct triosephosphate isomerase genes that provided different subunit species for the dimeric isoenzyme molecules. The 6 × wheats contained five, the 4 × wheats three, and the 2 × wheats only one chloroplastic isoenzyme band. The isoenzyme patterns were in accordance with a potential origin of one of the three chloroplastic triosephosphate isomerase genes of 6 × wheats from an Aegilops ancestor. The descent of the other two genes was, however, not in accordance with common contentions on the general evolution of cultural wheats. In the reciprocal intergeneric hybrids Secalotricum and Triticale both the chloroplastic and the cytosolic isoenzyme patterns of rye and wheat were biparentally inherited, indicating that both isoenzymes were controlled by nuclear genes. When monitored by immunoblotting the chloroplastic triosephosphate isomerase isoenzymes may provide useful genetic markers.     

Triosephosphate isomerases and aldolases from light- and dark-grown Euglena gracilis

Euglena gracilis synthesizes two distinct types of triosephosphate isomerase which can be resolved by isoelectric focusing. The more acidic Type A isomerase (pI = 4.4) predominates when cells are grown photoautotrophically and is localized in the chloroplasts. The Type B isoenzyme exhibits a more basic isoelectric pH (pI = 4.8), predominates under heterotrophic growth conditions and is of cytoplasmic origin. The two isoenzymes exhibit similar molecular weights (56,000–60,000) and catalytic properties but can be distinguished by their pH activity profiles. The situation parallels that of fructose diphosphate aldolase where a chloroplastic Class I enzyme (pI = 4.6, M_r 120,000) found in autotrophically grown cells can be resolved from the cytoplasmic Class II (pI = 5.7, M_r 88,000) enzyme which predominates under heterotrophic conditions. Inhibition of chloroplastic 70S ribosomal synthesis by chloramphenicol blocks the formation of the Type A triosephosphate isomerase and the Class I aldolase.     

Triosephosphate isomerase from young and old Turbatrix aceti.

Triosephosphate isomerase (EC 5.3.1.1) has been purified from young and old Tubatrix aceti. The enzyme shows a sharply lower specific activity in homogenates from old nematodes compared to similar preparations from young animals. However, when the enzyme activity of the respective homogenates was adjusted to the same level, equal amounts of antiserum (prepared to pure “young” triosephosphate isomerase) were required to remove the activity. Therefore, the lower specific activity observed in “old” homogenates was due to the presence of less enzyme and not to “altered” enzyme. The same results were obtained by immunotitrations of pure preparations of “young” and “old” enzyme. Moreover, in contrast to results reported for other enzymes, the specific activity of “old” triosephosphate isomerase, during purification, rose to the same value as that of pure “young” enzyme. The evidence indicates that altered triosephosphate isomerase does not exist in old T. aceti. The above results contradict the idea of an “error theory” in which all proteins would develop altered sequences. Pure triosephosphate isomerase (old or young) from T. aceti consists of two subunits, each of molecular weight 26,500. No isozymes could be detected.     

Repression of the Plastidic Isoenzymes of Aldolase, 3-Phosphoglycerate Kinase, and Triosephosphate Isomerase in the Barley Mutant "albostrians"

White leaves of the mutant line albostrians and green leaves of the wild-type cultivar Salome of barley (Hordeum vulgare L.) were screened for the presence of plastidic and cytosolic isoenzymes of sugar-phosphate metabolism. Isoenzyme separation was achieved by anion-exchange chromatography on Fractogel TSK DEAE-650(S). The mutant tissue had a markedly reduced level of plastidic 3-phosphoglycerate kinase, triosephosphate isomerase, and aldolase activity. In contrast, the activity of plastidic glucosephosphate isomerase, fructose 1,6-bisphosphatase, 6-phosphogluconate dehydrogenase, starch phosphorylase, and ADP-glucose pyrophosphorylase was in the same range as in wild-type leaf tissue. The activity of the corresponding cytosolic isoenzymes (including UDP-glucose pyrophosphorylase) showed essentially no differences in mutant and wild type. The same trend was observed in dark-grown mutant and wild-type leaves. Interestingly, the total activity levels of all isoenzymes were about the same when comparing dark-grown and light-grown mutant or wild-type plants. From these data, it is concluded that mutant leaves exhibit a selective decrease of a subgroup of plastidic isoenzymes associated with the Calvin cycle.     

Conversion by trypsin of nonsense suppressor-produced isozymes of triosephosphate isomerase to isozymes resembling the wild type.

Nonsense suppressor genes caused the synthesis of new triosephosphate isomerase isozymes in Bacillus subtilis. Incubation with trypsin produced a large decrease in the apparent molecular weight of one such isozyme and simultaneously changed the electrophoretic behavior such that it resembled that of the wild-type enzyme.     

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase.

We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild-type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3-amino-acid hinges: X(A/S)(L/K/M), X(aromatic/beta-branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families.     

The structure of the thioredoxin-triosephosphate isomerase complex provides insights into the reversible glutathione-mediated regulation of triosephosphate isomerase

Protein-protein interactions are crucial for many biological functions. The redox interactome encompasses numerous weak transient interactions in which thioredoxin plays a central role. Proteomic studies have shown that thioredoxin binds to numerous proteins belonging to various cellular processes, including energy metabolism. Thioredoxin has cross talk with other redox mechanisms involving glutathionylation and has functional overlap with glutaredoxin in deglutathionylation reactions. In this study, we have explored the structural and biochemical interactions of thioredoxin with the glycolytic enzyme, triosephosphate isomerase. Nuclear magnetic resonance chemical shift mapping methods and molecular dynamics-based docking have been applied in deriving a structural model of the thioredoxin-triosephosphate isomerase complex. The spatial proximity of active site cysteine residues of thioredoxin to reactive thiol groups on triosephosphate isomerase provides a direct link to the observed deglutathionylation of cysteine 217 in triosephosphate isomerase, thereby reversing the inhibitory effect of S-glutathionylation of triosephosphate isomerase.     

1H nuclear magnetic resonance assay of erythrocyte triosephosphate isomerase

A direct method for measuring the activity of erythrocyte triosephosphate isomerase using 1H NMR spectroscopy was developed. NMR spectroscopy allows the simultaneous monitoring of the substrate and the product of the reaction by virtue of the differences in the NMR spectrum of each chemical species. The assay conditions were based on a modification of a conventional spectrophotometric method. The enzymatic activity measured using NMR gave results comparable to those obtained in a standard assay. The results were used in the kinetic characterization of triosephosphate isomerase in hemolysates from subjects with homozygous or heterozygous deficiency of the enzyme. In general, NMR spectroscopy has the potential for wide application in the rapid development of new enzyme assays.     

Inhibition of triosephosphate isomerase from Trypanosoma brucei with cyclic hexapeptides.

Two series of oligopeptides have been synthesized. Their effects on the activity of purified triosephosphate isomerase from Trypanosoma brucei and various other organisms have been studied. Using detailed three-dimensional structure information, the first series consisted of both cyclic and linear hydrophilic peptides that were designed to mimic the beta turns of the subunit interface loops of the trypanosome triosephosphate isomerase dimer. None of these exerted any inhibitory effect. The second series consisted of more hydrophobic cyclic peptides, originally designed to inhibit a hepatic transport system. Several of these were very effective in inhibiting the trypanosome triosephosphate isomerase, but not the homologous enzymes from rabbit, dog, yeast or Escherichia coli. The most active peptide, cyclo[-Trp-Phe-D-Pro-Phe-Phe-Lys(Z)-], exerted 50% inhibitory activity at a concentration of 3 microM. The nature of the inhibitory action of one of these compounds cyclo[-Trp-Tyr(OSO3Na)-D-Pro-Phe-Thr(OSO3Na)-Lys(Z)-] was studied in more detail. Its inhibition was noncompetitive and reversible and more than one peptide was able to bind/active site.     

Structure determination of the glycosomal triosephosphate isomerase from Trypanosoma brucei brucei at 2.4 A resolution

The three-dimensional crystal structure of the enzyme triosephosphate isomerase from the unicellular tropical blood parasite Trypanosoma brucei brucei has been determined at 2.4 A resolution. This triosephosphate isomerase is sequestered in the glycosome, a unique trypanosomal microbody of vital importance for the energy-generating machinery of the trypanosome. The crystals contain one dimer per asymmetric unit. The structure could be solved by the method of molecular replacement, using the refined co-ordinates of chicken triosephosphate isomerase as a search model. The positions and individual isotropic temperature factors of the 3792 atoms of the complete dimer have been refined by the Hendrickson & Konnert restrained refinement procedure. While tight restraints have been maintained on the bonded distances, the R-factor has dropped to 23.2% for 12317 reflections between 6 A and 2.4 A. A total of 0.6 mg of enzyme was used for establishing the correct crystallization conditions and solving the three-dimensional structure. Although the sequences of trypanosomal and chicken triosephosphate isomerase are identical at only 52% of the 247 common positions, the overall folds are very similar. The architecture of the active sites is virtually the same with 85% of the side-chains being identical. On the other hand, the residues involved in the dimer contacts are the same at only 55% of the positions. Nevertheless, the position of the local 2-fold axis in the chicken and glycosomal dimers is similar. A remarkable feature of glycosomal triosephosphate isomerase is its high overall positive charge. This extra charge is concentrated in four clusters of positively charged side-chains on the surface of the dimer, quite far away from the active site. These clusters may be involved in the mechanism of import of this triosephosphate isomerase into the glycosome.     

The adaptability of the active site of trypanosomal triosephosphate isomerase as observed in the crystal structures of three different complexes

Crystals of triosephosphate isomerase from Trypanosoma brucei brucei have been used in binding studies with three competitive inhibitors of the enzyme's activity. Highly refined structures have been deduced for the complexes between trypanosomal triosephosphate isomerase and a substrate analogue (glycerol-3-phosphate to 2.2 A), a transition state analogue (3-phosphonopropionic acid to 2.6 A), and a compound structurally related to both (3-phosphoglycerate to 2.2 A). The active site structures of these complexes were compared with each other, and with two previously determined structures of triosephosphate isomerase either free from inhibitor or complexed with sulfate. The comparison reveals three conformations available to the "flexible loop" near the active site of triosephosphate isomerase: open (no ligand), almost closed (sulfate), and fully closed (phosphate/phosphonate complexes). Also seen to be sensitive to the nature of the active site ligand is the catalytic residue Glu-167. The side chain of this residue occupies one of two discrete conformations in each of the structures so far observed. A "swung out" conformation unsuitable for catalysis is observed when sulfate, 3-phosphoglycerate, or no ligand is bound, while a "swung in" conformation ideal for catalysis is observed in the complexes with glycerol-3-phosphate or 3-phosphonopropionate. The water structure of the active site is different in all five structures. The results are discussed with respect to the triosephosphate isomerase structure function relationship, and with respect to an on-going drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.     

The influence of pH on the interaction of inhibitors with triosephosphate isomerase and determination of the pKa of the active-site carboxyl group.

Ionization effects on the binding of the potential transition state analogues 2-phosphoglycolate and 2-phosphoglycolohydroxamate appear to be attributable to the changing state of ionization of the ligands themselves, therefore it is unnecessary to postulate the additional involvement of an ionizing residue at the active site of triosephosphate isomerase to explain the influence of changing pH on Ki in the neutral range. The binding of the competitive inhibitor inorganic sulfate is insensitive to changing pH in the neutral range. 3-Chloroacetol sulfate, synthesized as an active-site-specific reagent for triosephosphate isomerase, is used to provide an indication of the pKa of the essential carboxyl group of this enzyme. Previously described active-site-specific reagents for the isomerase were phosphate esters, and their changing state of ionization (accompanied by possible changes in their affinity for the active site) may have complicated earlier attempts to determine the pKa of the essential carboxyl group from the pH dependence of the rate of inactivation. Being a strong monoprotic acid, chloroacetol sulfate is better suited to the determination of the pKa of the carboxyl group. Chloroacetol sulfate inactivates triosephosphate isomerase by the selective esterification of the same carboxyl group as that which is esterified by the phosphate esters described earlier. From the pH dependence of the rate of inactivation of yeast triosephosphate isomerase, the apparent pKa of the active-site carboxyl group is estimated as 3.9 +/- 0.1.     

Synthesis of the pentadecapeptide sequence of the active site of rabbit muscle triosephosphate isomerase.

The pentadecapeptide fragment, Trp-Val-Leu-Ala-Tyr-Glu-Pro-Val-Trp-Ala-Ile-Gly-Thr-Gly-Lys, which constitutes a part of the active site of rabbit muscle triosephosphate isomerase has been synthesized. It does not exhibit any catalytic activity typical of triosephosphate isomerase.     

Structural considerations for the rational design of selective anti-trypanosomal agents: the role of the aromatic clusters at the interface of triosephosphate isomerase dimer

Seven benzothiazoles were successfully docked into the interface of both human and trypanosomal triosephosphate isomerases, and the binding free energies of each complex were calculated using the program AutoDock. Structural and energetical analysis of the complexes showed that large benzothiazoles could form more stable complexes with trypanosomal triosephosphate isomerase than with human triosephosphate isomerase. Thus, we hypothesize that the distribution of the residues forming the aromatic clusters at the enzyme's interface and the size of the inhibitors might play a crucial role in the selective inhibition of TcTIM. Following the findings here presented, it is possible to better determine the structural elements involved in the origin of the selectivity at the trypanosomal triosephosphate isomerase interface, and to enable efficient anti-trypanosomal drug design strategies.     

The hyperthermophilic bacterium Thermotoga neapolitana possesses two isozymes of the 3-phosphoglycerate kinase/triosephosphate isomerase fusion protein

Abstract The phosphoglycerate kinase ( pgk ), triosephosphate isomerase ( tpi ), and enolase ( eno ) genes from Thermotoga neapolitana have been cloned and expressed in Escherichia coli . In high copy number, the pgk gene complemented an E. coli pgk ⁻ strain. In T. neapolitana , the pgk and tpi genes appear to be fused and eno is near those genes. Like T. maritima , T. neapolitana produces phosphoglycerate kinase as both an individual enzyme and a fusion protein with triosephosphate isomerase, and triosephosphate isomerase activity is not found without associated phosphoglycerate kinase activity. Unlike T. maritima , which forms only a 70-kDa fusion protein, T. neapolitana expresses both 73-kDa and 81-kDa isozymes of this fusion protein. These isozymes are present in both T. neapolitana cells and in E. coli cells expressing T. neapolitana genes.     

Characterization of three electrophoretic variants of human erythrocyte triosephosphate isomerase found in Japanese

Three new electrophoretic variants of human erythrocyte triosephosphate isomerase (TPI) have been partially purified and compared with the normal isozyme with respect to stability, kinetics, and immunological properties. TPI 2HR1, an anodally migrating variant, was less stable than normal in guanidine denaturation and thermodenaturation tests, although it exhibited normal kinetic properties. The level of enzyme activity in erythrocytes from the proband with the phenotype TPI 1-2HR1 was about 60% of the normal mean. The variant allozyme TPI 2NG1, an anodally migrating allozyme associated with normal activity, was very thermolabile at 55 and 57°C. It was also much more labile than normal in stability tests in buffers at pH 5 and pH 10, although it exhibited normal kinetic and immunological properties. TPI 4NG1, a cathodally migrating variant associated with normal activity and normal kinetic as well as immunological properties, was more stable than normal in pH 5 buffer. Family studies demonstrated that the unique characteristics of these variants are genetically transmitted. In two-dimensional electrophoresis of purified isozymes the variant subunits were separated from the normal in the pI axis. However, there is no difference between the variants and the normal in the molecular weight axis, suggesting that the variants result from single amino acid substitutions.     

Separation of E. coli tRNA-Tyr precursor RNA from E. coli tRNA by hydroxyapatite chromatography

An assay procedure is described for triosephosphate isomerase based on measurement of the ellipticity of l-glyceraldehyde 3-phosphate remaining when d,l-glyceraldehyde 3-phosphate is the source of substrate and d-glyceraldehyde 3-phosphate is converted by triosephosphate isomerase to dihydroxyacetone phosphate. The assay method has advantages over the conventional coupled-enzyme assays in that it circumvents the difficulties posed by instability of the coupling enzymes and their cofactors, as well as by inhibitors of triosephosphate isomerase which may be present in preparations of the coupling enzymes. Although the method is not suited for routine assays during purification or in most clinical applications, it has advantages for detailed kinetic studies where pH, temperature, or other factors cause the coupled-enzyme assay procedures to be unreliable.     

Structure and metabolism of mammalian liver glycogen monitored by carbon-13 nuclear magnetic resonance

Natural-abundance 13C NMR signals from glycogen are observable in situ within the perfused livers of rats. The nuclear magnetic relaxation properties (T1, T2, eta + 1) of glycogen were measured for glycogen in situ and in vitro and were found to be identical. All of the carbon nuclei in glycogen contribute to the high-resolution NMR spectrum, in spite of glycogen's very large molecular weight. The metabolism of glycogen in situ in the perfused rat liver was followed by 13C NMR. Stimulation of the fed rat liver by physiological glucagon levels led to rapid glycogenolysis. Perfusion of the liver with [1-13C]glucose led to net glycolysis, with concomitant scrambling of the label from C1 to C6 due to triosephosphate isomerase activity.     

Fructose metabolizing enzymes in the rat liver and metabolic parameters: Interactions between dietary copper,type of carbohydrates,and gender

This study was conducted to determine the effects of nutrient interactions between dietary carbohydrates and copper levels on fructose-metabolizing hepatic enzymes in male and female rats. Male and female rats were fed diets for 5 weeks that were either adequate or deficient in copper that contained either starch or fructose. Rats of both sexes fed fructose as compared with those fed starch showed higher activity of hepatic fructose metabolizing enzymes. There were also significant differences in fructose metabolism of liver between the male and female rats. Female rats had lower hepatic ketohexokinase and triose kinase but higher triosephosphate isomerase activities compared with male rats. Male rats fed copper-deficient diets had lower aldolase B activity compared with those fed copper-adequate diets. Female rats fed copper-deficient diets had higher triosephosphate isomerase activity compared with rats fed copper-adequate diets. Our data suggest that gender differences in hepatic fructose metabolism may not be the primary reason for the severity of copper deficiency syndrome in male rats fed copper-deficient diet with fructose.