Human triosephosphate isomerase cDNA and protein structure. Studies of triosephosphate isomerase deficiency in man

Nine cDNA clones of human adult liver triosephosphate (TP) isomerase have been isolated and characterized. All nine appear to be derived from a single mRNA species. DNA sequencing of one clone, designated pHTPI-5a, defined the last two nucleotides of the methionine initiation codon, the entire 744-nucleotide coding region of the mature polypeptide, and the entire 448-nucleotide 3' untranslated region. The frequency of TP isomerase clones in the cDNA library suggests that TP isomerase mRNA is present in adult liver at approximately 25 copies/cell. A single, low abundance TP isomerase mRNA species was detected in RNA isolated from normal human fibroblast cell lines. Analysis of TP isomerase mRNA levels in cultured fibroblasts of individuals that are homozygous for TP isomerase deficiency revealed normal levels in one and approximately 40% of normal levels in another. From this small patient sampling, it can be concluded that the genetic basis for TP isomerase deficiency is heterogeneous.     

Drosophila model of human inherited triosephosphate isomerase deficiency glycolytic enzymopathy

Heritable mutations, known as inborn errors of metabolism, cause numerous devastating human diseases, typically as a result of a deficiency in essential metabolic products or the accumulation of toxic intermediates. We have isolated a missense mutation in the Drosophila sugarkill (sgk) gene that causes phenotypes analogous to symptoms of triosephosphate isomerase (TPI) deficiency, a human familial disease, characterized by anaerobic metabolic dysfunction resulting from pathological missense mutations affecting the encoded TPI protein. In Drosophila, the sgk gene encodes the glycolytic enzyme TPI. Our analysis of sgk mutants revealed TPI impairment associated with reduced longevity, progressive locomotor deficiency, and neural degeneration. Biochemical studies demonstrate that mutation of this glycolytic enzyme gene does not result in a bioenergetic deficit, suggesting an alternate cause of enzymopathy associated with TPI impairment.     

Human triosephosphate isomerase deficiency resulting from mutation of Phe-240.

Triosephosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketolisomerase [E.C.5.3.1.1]) deficiency is an autosomal recessive disorder that typically results in chronic, nonspherocytic hemolytic anemia and in neuromuscular impairment. The molecular basis of this disease was analyzed for one Hungarian family and for two Australian families by localizing the defects in TPI cDNA and by determining how each defect affects TPI gene expression. The Hungarian family is noteworthy in having the first reported case of an individual, A. Jó., who harbors two defective TPI alleles but who does not manifest neuromuscular disabilities. This family was characterized by two mutations that have never been described. One is a missense mutation within codon 240 (TTC [Phe]-->CTC [Leu]), which creates a thermolabile protein, as indicated by the results of enzyme activity assays using cell extracts. This substitution, which changes a phylogenetically conserved amino acid, may affect enzyme activity by disrupting intersubunit contacts or substrate binding, as deduced from enzyme structural studies. The other mutation has yet to be localized but reduces the abundance of TPI mRNA 10-20-fold. Each of the Australian families was characterized by a previously described mutation within codon 104 (GAG [Glu]-->GAC [Asp]), which also results in thermolabile protein.     

Increased formation of methylglyoxal and protein glycation, oxidation and nitrosation in triosephosphate isomerase deficiency

Triosephosphate isomerase deficiency is associated with the accumulation of dihydroxyacetonephosphate (DHAP) to abnormally high levels, congenital haemolytic anaemia and a clinical syndrome of progressive neuromuscular degeneration leading to infant mortality. DHAP degrades spontaneously to methylglyoxal (MG)--a potent precursor of advanced glycation endproducts (AGEs). MG is detoxified to D-lactate intracellularly by the glyoxalase system. We investigated the changes in MG metabolism and markers of protein glycation, oxidation and nitrosation in a Hungarian family with two germline identical brothers, compound heterozygotes for triosephosphate isomerase deficiency, one with clinical manifestations of chronic neurodegeneration and the other neurologically intact. The concentration of MG and activity of glyoxalase I in red blood cells (RBCs) were increased, and the concentrations of D-lactate in blood plasma and D-lactate urinary excretion were also increased markedly in the propositus. There were concomitant increases in MG-derived AGEs and the oxidative marker dityrosine in hemoglobin. Smaller and nonsignificant increases were found in the neurologically unaffected brother and parents. There was a marked increase (15-fold) in urinary excretion of the nitrosative stress marker 3-nitrotyrosine in the propositus. The increased derangement of MG metabolism and associated glycation, oxidative and nitrosative stress in the propositus may be linked to neurodegenerative process in triosephosphate isomerase deficiency.     

Primary structure of human triosephosphate isomerase

Human placental triosephosphate isomerase was isolated by an improved procedure and recovered with the highest specific activity ever reported. Employing this purification procedure, sufficient amounts of the enzyme were obtained for detailed primary structural studies. For sequences analysis, the enzyme was reduced and carboxymethylated and subjected to tryptic and chymotryptic digestions. The peptide mixtures were separated by high-performance liquid chromatography using octyl or alkylphenyl reverse-phase columns and trifluoroacetic acid/acetonitrile gradient elution systems. Sequence analyses of the intact enzyme, tryptic, chymotryptic, and cyanogen bromide peptides were accomplished using high-sensitivity solid-phase sequencing procedures with either 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate or phenylisothiocyanate. The primary structure of human triosephosphate isomerase is constructed from the alignment of the tryptic peptides with the analysis of the overlapping chymotryptic peptides. The enzyme is a dimeric molecule consisting of two identical polypeptide chains with 248 amino acid residues and a calculated subunit molecular mass of 26,750 daltons. A comparison of the amino acid sequences from the human placental enzyme and from other species such as rabbit, chicken, and coelacanth muscles showed relatively high sequence homology, indicating that the evolution of the enzyme is very conservative. The amino acids of the active-site pocket and the subunit-subunit contact sites exhibit few changes.     

In vitro deamidation of human triosephosphate isomerase

The effects of pH, temperature, buffer ion, ionic strength, protein concentration, and substrate on the rates of specific, spontaneous deamidations of Asn-15 and Asn-71 of human triosephosphate isomerase were examined. Elevated temperature and pH facilitate the deamidations, and the deamidation rate is dependent on the specific buffer ions indicating a general base catalysis mechanism. The presence of substrate also enhances the rates of deamidation. The effect of substrate may be related to conformational changes in the catalytic center which are known to cause changes in the subunit-subunit contact sites where Asn-15 and Asn-71 are located. The enhanced deamidation in the presence of substrate may, in part, account for the more rapid rate of deamidation observed in vivo.     

Dimerization affects collective dynamics of triosephosphate isomerase

Molecular dynamics simulations (30-60 ns runs) are performed on free/apo triosephosphate isomerase (TIM) to determine any correlation between collective motions and loop 6 dynamics. Native TIM is reported to be active only as a homodimer even though cooperativity has not been observed between the two identical subunits. Both dimeric and monomeric (isolated from dimer) forms of TIM are simulated in explicit water at 300 K and 1 bar to inspect any differences between the structures in terms of fluctuation dynamics and functionally important loop 6 dynamics/closure. Significant cross-correlations between residue fluctuations are observed in the dimer, which result from the global counter-rotations of the two identical subunits in the essential modes of the dimer. Specifically, the first essential mode contributing to 34% of overall motion of the dimer is strongly coupled to the loop 6's closure over the active site. In contrast, such significant correlations cannot be observed in the monomeric structure, which maintains relatively localized motions of the loops in the essential modes. Thus, the onset of collective motions at ns time scale due to dimerization has functional implications as to the coordination of loop 6 closure.     

Computational modeling of the catalytic reaction in triosephosphate isomerase

We present a comprehensive analysis of the catalytic cycle of the enzyme triosephosphate isomerase (TIM), including both the reactive chemistry and the catalytic loop and side-chain motions. Combining accurate mixed quantum mechanics/molecular mechanics (QM/MM) and protein structure prediction methods, we have modeled both the structural and chemical aspects of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to d-glyceraldehyde 3-phosphate (GAP), for which there is a wealth of experimental data. The conjunction of this novel computational approach with the use of the recent near-atomic resolution TIM-DHAP Michaelis complex PDB structure, 1NEY.pdb, has enabled us to obtain robust qualitative and, where available, quantitative agreement with a wide range of experimental data. Among the principal conclusions that we are able to draw are the importance of the monoanionic (as opposed to dianioic) form of the substrate phosphate group in the catalytic cycle, detailed positioning and energetics of the key catalytic residues in the active-site, the flexible nature of Glu165, which favors its direct involvement in the formation of the enediol intermediate, energetics of the open and closed form of the catalytic loop region in the presence and absence of substrate, and quantitative reproduction of various experimentally measured reaction rates, typically to within approximately 1 kcal/mol. Our results are consistent with the available experimental data, and provide an initial picture as to why loop opening when GAP is the product has a higher barrier than when DHAP is the product.     

Proton diffusion in the active site of triosephosphate isomerase

The current model for hydrogen flow in the aldose-ketose isomerases is probably incorrect. Enzymes of this class are characterized by both hydrogen transfer and proton exchange in the interconversion of substrate and product. The transfer is believed to be due to the action of a unique basic residue in the active site. Exchange is presumed to occur by dissociation of the abstracted proton and reassociation from the medium prior to its transfer to the intermediate enediol on the way to product. Dissociation of a necessary proton from the intermediate state imposes limits on the overall catalytic rate depending on the pKa of the protonated base and the pH of the medium. A case in point is triose-P isomerase (TIM), where kcat is approximately 10(4) s-1. T-Labeled substrate is found to lose approximately 95% of its T to the medium when totally converted to product. Although the active site base is believed to be a glutamate of pKa = 3.9, the pH dependence of maximum velocity is known to be flat up to pH 10. The loss of hydrogen required to form product as indicated by isotope exchange must be restored completely at this high pH, requiring a base of very high pKa, or there must be some other explanation for the loss of isotope. The present study demonstrates the existence of a single proton on human and rabbit TIM and three protons on yeast TIM that rapidly exchange with the abstracted proton at the E.enediol state internal exchange. Exchange with the medium external exchange occurs from the enzyme after substrate or product has dissociated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevalence of partial deficiency of red cell triosephosphate isomerase in Germany — a study of 3000 people

Summary During a heterozygote screening of nearly 3000 persons, triosephosphate isomerase (TPI) deficiencies in erythrocytes were discovered in 11 unrelated persons, showing a residual activity between 39 and 76% of normal activity. Extensive genealogic studies were performed to confirm that these persons with TPI deficiency were heterozygous carriers. The total heterozygote frequency of triosephosphate isomerase deficiencies was 3.7/1000.The persons with heterozygous deficiency could be divided into two categories. Subjects of category I had a mean residual activity of 49% of the expected normal activity and were represented by a frequency of 1.3/1000. Subjects of category II had a mean residual activity of 67% of the expected normal activity and were represented by a frequency of 2.4/1000. None of the heterozygous persons showed an electrophoretic variant. The immunologic specific activity was normal with one exception. Therefore, we assume that in many cases of our heterozygous TPI-deficiencies a TPI protein with a normal specific activity is synthesized to a diminished degree.     

The denaturation-renaturation of chicken-muscle triosephosphate isomerase in guanidinium chloride

1. The process of denaturation of the chicken muscle dimeric enzyme triosephosphate isomerase on addition of guanidinium chloride has been studied at pH 7.6, the pH at which the recovery of activity is optimal (100%) on removal of denaturant. Determinations of the sedimentation coefficient, intrinsic viscosity, molecular weight (by sedimentation equilibrium studies) and the absorption coefficient at 280 nm in various concentrations of guanidinium chloride concurred in showing a single, sharp transition at about 0.7 M guanidinium chloride at a protein concentration 1-5 mg/ml from the native enzyme to the dissociated, unfolded chains of the monomer. Relative fluorescent intensity measurements revealed a single transition at about 0.4 M guanidinium chloride at enzyme concentrations of about 0.05 mg/ml. 2. The process of denaturation in different guanidinium chloride concentrations was first order with respect to enzyme and about sixth order with respect to denaturant. 3. The rate of attainment of equilibrium during the renaturation obeyed second-order/first-order reversible kinetics. It was concluded that the rate-determining step in renaturation at pH 7.6 must be the association of two subunits.     

Antibodies to triosephosphate isomerase in patients with neuropsychiatric lupus

Sera from patients with neuropsychiatric lupus (NP lupus) were screened for antibodies to mouse choroid plexus cell line ECPC-4 by Western blotting. A 29-kDa protein band detected in NP lupus sera was identified as triosephosphate isomerase (TPI). Using Western blotting with TPI, TPI was confirmed as the reactive molecule in sera (6 of 14 samples) and in cerebrospinal fluids (1 of 2 samples) of patients with NP lupus. Enzyme-linked immunosorbent assay with TPI showed that the serum anti-TPI antibody index of 89.8 (SD, 70.1) in NP lupus group was significantly higher than in systemic lupus erythematosus without NP manifestations, 34.6 (29.6); scleroderma, 38.2 (39.9); polymyositis/dermatomyositis, 42.1 (51.5); and control, 31.7 (27.4) groups (p<0.02). Sensitivity, specificity, and positive and negative likelihood ratios of serum anti-TPI antibody index for NP lupus were 42.9%, 94.7%, 8.1%, and 0.6%, respectively. These results suggest that anti-TPI antibodies are closely associated with NP lupus.