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.     

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.     

Control of the reactivation kinetics of homodimeric triosephosphate isomerase from unfolded monomers

Homodimeric triosephosphate isomerases from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar catalytic properties and 3-D structures; their overall amino acid sequence identity is 68% and 85% in their interface residues. Nonetheless, active dimer formation from guanidinium chloride unfolded monomers is faster and more efficient in TcTIM than in TbTIM. The enzymes thus provide a unique opportunity for exploring the factors that control the formation of active dimers. The kinetics of reactivation at different protein concentrations showed that the process involved three reactions: monomer folding, association of folded monomers, and a transition from inactive to active dimers. The rate constants of the reactions indicated that, at relatively low protein concentrations, the rate-limiting step of reactivation was the association reaction; at high protein concentrations the transition of inactive to active dimers was rate limiting. The rates of the latter two reactions were higher in TcTIM than in TbTIM. Studies with a mutant of TcTIM that had the interface residues of TbTIM showed that the association rate constant was similar to that of TbTIM. However, the rate of the transition from inactive to active dimers was close to that of TcTIM; thus, this transition depends on the noninterfacial portion of the enzymes. When unfolded monomers of TcTIM and TbTIM were allowed to reactivate together, TcTIM, the hybrid, and TbTIM were formed in a proportion of 1:0.9:0.2. This distribution suggests that, in the hybrid, the characteristics of the TcTIM monomers influence the properties of TbTIM monomers.     

Cytosolic triosephosphate isomerase is a single gene in rice.

A cDNA clone encoding rice (Oryza sativa L.) cytosolic triosephosphate isomerase (TPI), an important glycolytic enzyme, was isolated and characterized. The clone (pRTPI-6) contains an open reading frame of 759 base pairs, encoding a polypeptide chain of 253 amino acid residues (M(r) 27,060). The identity of this clone was defined by its high homology (85% nucleotide sequence and 89% amino acid sequence identical match) with a maize mRNA sequence encoding the cytosolic TPI and with TPIs from other species. Genomic DNA blot analysis using the cDNA as a probe showed that the cytosolic TPI gene is present as a single copy per haploid rice genome, as opposed to that found in maize, in which multiple TPI gene copies exist. A single TPI mRNA species of about 1100 nucleotides was detected by gel blot hybridization analysis of RNA isolated from root, culm, and leaf tissues, indicating that its expression is ubiquitous. Based on sequence comparison and molecular analysis, we propose that the chloroplast-located TPI may be encoded by divergent structural nuclear genes in rice.     

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.     

Premature translation termination mediates triosephosphate isomerase mRNA degradation.

We characterized an anemia-inducing mutation in the human gene for triosephosphate isomerase (TPI) that resulted in the production of prematurely terminated protein and mRNA with a reduced cytoplasmic half-life. The mutation converted a CGA arginine codon to a TGA nonsense codon and generated a protein of 188 amino acids, instead of the usual 248 amino acids. To determine how mRNA primary structure and translation influence mRNA stability, in vitro-mutagenized TPI alleles were introduced into cultured L cells and analyzed for their effect on TPI RNA metabolism. Results indicated that mRNA stability is decreased by all nonsense and frameshift mutations. To determine the relative contribution of the changes in mRNA structure and translation to the altered half-life, the effects of individual mutations were compared with the effects of second-site reversions that restored translation termination to normal. All mutations that resulted in premature translation termination reduced the mRNA half-life solely or mainly by altering the length of the mRNA that was translated. The only mutation that altered translation termination and that reduced the mRNA half-life mainly by affecting the mRNA structure was an insertion that shifted termination to a position downstream of the normal stop codon.     

Structural determinants for ligand binding and catalysis of triosephosphate isomerase.

The crystal structure of leishmania triosephosphate isomerase (TIM) complexed with 2-(N-formyl-N-hydroxy)-aminoethyl phosphonate (IPP) highlights the importance of Asn11 for binding and catalysis. IPP is an analogue of the substrate D-glyceraldehyde-3-phosphate, and it is observed to bind with its aldehyde oxygen in an oxyanion hole formed by ND2 of Asn11 and NE2 of His95. Comparison of the mode of binding of IPP and the transition state analogue phosphoglycolohydroxamate (PGH) suggests that the Glu167 side chain, as well as the triose part of the substrate, adopt different conformations as the catalysed reaction proceeds. Comparison of the TIM-IPP and the TIM-PGH structures with other liganded and unliganded structures also highlights the conformational flexibility of the ligand and the active site, as well as the conserved mode of ligand binding.     

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.     

Critical ionization states in the reaction catalyzed by triosephosphate isomerase.

To allow the detailed interpretation of the pH dependences of the steady-state parameters for the reaction catalyzed by triosephosphate isomerase, three kinds of experiments have been performed. First, the value of kcat/Km for enzyme-catalyzed isomerization of the phosphonate analogue of D-glyceraldehyde 3-phosphate (2-hydroxy-4-phosphonobutyraldehyde) has been shown to titrate with an apparent pKa of 7.5, which is close to the phosphonate's second ionization constant. Secondly, the sulfate ester analogue of dihydroxyacetone phosphate (dihydroxyacetone sulfate), which exists only as a monoanion over the pH range of interest, has been shown not to bind detectably to the enzyme. Thirdly, an isotopic discrimination experiment at pH 5.2 has been compared with a similar investigation at pH 7.6. The results together demonstrate that both enzyme and substrate ionizations control the reaction rate in the pH range 5 to 8.     

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.     

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)     

Structure and inactivation of triosephosphate isomerase from Entamoeba histolytica

Triosephosphate isomerase (TIM) has been proposed as a target for drug design. TIMs from several parasites have a cysteine residue at the dimer interface, whose derivatization with thiol-specific reagents induces enzyme inactivation and aggregation. TIMs lacking this residue, such as human TIM, are less affected. TIM from Entamoeba histolytica (EhTIM) has the interface cysteine residue and presents more than ten insertions when compared with the enzyme from other pathogens. To gain further insight into the role that interface residues play in the stability and reactivity of these enzymes, we determined the high-resolution structure and characterized the effect of methylmethane thiosulfonate (MMTS) on the activity and conformational properties of EhTIM. The structure of this enzyme was determined at 1.5A resolution using molecular replacement, observing that the dimer is not symmetric. EhTIM is completely inactivated by MMTS, and dissociated into stable monomers that possess considerable secondary structure. Structural and spectroscopic analysis of EhTIM and comparison with TIMs from other pathogens reveal that conformational rearrangements of the interface after dissociation, as well as intramonomeric contacts formed by the inserted residues, may contribute to the unusual stability of the derivatized EhTIM monomer.