TRIOSEPHOSPHATE ISOMERASE (TPI) A DIMERIC GLYCOLYTIC ENZYME AS A MODEL OF TIM-BARREL ACTIVE-SITE STRUCTURAL AND CHEMICAL ASPECTS IN THE MONOMER LOOP REGION'S REVERSIBLE CATALYTIC REACTION.

Triosephosphate isomerase (TPI, EC 5.3.1.1) (§, ‡) is essential to glycolysis, catalyzes the fifth step in the glycolysis pathway the reversible conversion of dihydroxyacetone phosphate (DHAP) into glyceraldehyde-3-phosphate. TPI is a homodimer formed by two identical dimeric molecules of a single structural locus : 12p13.31. TPI has only 1 functional gene with a molecular mass of 29 kDa, that after refinement are products of a distinct single structural locus. The variant phenotype of identical subunits are expressed in both red cells and circulating lymphocytes, catalyzing the interconversion of one of the two products breakdown by reversible conversion. The TPI substrate by deprotonation the transition state reaction of dihydroxyacetone phosphate (DHAP) substrate yields one product of the glycolytic pathway, is a trend* (Kcat) that persists creating the initial complex microcompartmentation of TPI to give (G3P) glyceraldehyde-3-phosphate which seems to be the isomerase* activity, release is slower than its conversion to DHAP in normal and TPI deficient cells. TIM with its natural substrates has not been (•) crystalized**. TPI is a dimeric enzyme and contains 7 exons interrupted by six introns.

The crystallographic structure of (HsTPI) human triosephosphate isomerase PDB:1HTI is one dimer per asymmetric unit subunit 1 and subunit 2 are in the open and closed conformations in the 3-dimensional asymmetric space group P 2(1) which is specific to the Monoclinic with minimization on the entire structure in the presence of substrate analogues and its surrounding residues supporting possible regions targeted for drug design.

TPI deficiency (TPID) a disorder of glycolysis, occurring in haplotypes of specific alleles heterogeneous to clinical TPI-deficiency, with a rare homozygous deficiency the resulting genetic defect is the cause of a null variant incompatible with life by abnormally high levels of DHAP which degrades spontaneously into the toxic (MG) methylglyoxal, due to deamidation of asparagine (Asn15-71) to form aspartic and glutamic acid. Loop 6 plays a role in preventing the breakdown yield of methylglyoxal (fMG) one of the of the three products of enzyme-bound enediol(ate) phosphate, towards elimination of (fMG) inorganic phosphate. TPI deficiency is due to the common aberrant dimerization (or the dissociation into inactive monomers) of mutation TPI 1591C, encoding a Glu104-to-Asp (glutamate-to-aspartate) substitution in the TPI variant found in cases of hemolytic anemia coupled with neurodegeneration, the Glu104-to-Asp substitution is the most common disease allele inherited, when compared to wild-type TPI's three (residues from the same subunit) similar but not identical interactions between the inhibitor and catalytic residues, Glu 167 (or 165) forms a stable dimer and provides the rationale for production of structurally normal enzyme in humans, the E104D mutation, provides the amyloid-resistant structure of human triosephosphate isomerase (HsTPI). Water-protein molecules join two catalytically active monomers which is only in its dimeric form, as monomers of TIM are not functional. Within a hydrophobic catalytic pocket of the native enzymes the binding and catalysis of TPIs in hemolysates, bind to the red cell membrane. Molecular modeling using the human crystal structure of TPI was performed to determine how these mutations could affect enzyme structure and function. The Amyloid secondary structure autoepitopes antigen-driven mechanism works toward recovery of the anti-triosephosphate isomerase mutant TPI peptide** antigens. This is the scheme that allows function-enhancing stability most significantly, the catalysis for deprotonation of DHAP or vice-versa GAP substrates of the TIM-barrel relative to TPI toward turnover of two-part substrate glycolaldehyde / phosphite dianion {GA + HPO32* the transition state for this enolising enzyme substrate pieces.} Km/obsd* group of the whole GAP substrate and H95 (loop 4) is also optimal for small mutational changes in or reflects its compatibility with amino acid residues which stabilizes the enediolate intermediate (GA/HPO) activity from change in the products scheme (a proton transfer mechanism) DHAP/G3P or interconversion of these intermediates.

Closed (activated for catalysis) of optimal WT (TPI) molecular modeling PDB 1HTI_B using the human crystal structure of TPI human triosephosphate isomerase (HsTPI) conformation 1hti_b, calculated to the incidence residue Water-protein molecules and the protein cage that interacts within a hydrophobic catalytic pocket isolated and examined which coded for human triose-phosphate isomerase. [EC: 5.3.1.1]….

The active flexible site loop must open before product release, unliganded in trypanosomal Tb-TIM glycerol phosphate ester to liganded Glu167 in the catalytic cycle and the enzymes substrate transition state between open and closed to protect the substrate for the turnover of DHAP and G3P (GAP) the natural substrates, and inhibiting the formation of a toxic by-product in the absence of this equilibration reactions between dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (G3P) enzymes by mutations that impair biosynthesis transforming competent cells, in the presence of an auxotrophic effect with these differences generated for an inability of the host organism to synthesize an essential compound during glycolysis in Tb-TIM. Trypanosomal-TIM is a glycolytic enzyme essential for the parasite survival that causes Chagas* disease, in this study G. bellum from the genus related Geraniaceae and its phenolic compound are leads which generates an unstable epimer of an enzyme Geranin A-containing changes resulting from ligand adducts in the active site to capture in addition a source of frustration that becomes more favourable. Glycolaldehyde (GA) the simplest sugar-related molecules uptake of a proton by Glu167 preserves the small effect for inhibition by PGA (transition-state analog) relative to substrate, G3P produces a triosephosphate isomerase with wild-type activity, loop 6 adopts the "closed" desolvated  (+) conformation to facilitate completion of catalysis by the formation of the › Michaelis-Menten complex (on the ‹ micros-ms › time scale) utilization yields further corrected calculations with corresponding (slower Kcat) motional rates* Km. Increase's are discussed in the context of the significance (Enzyme kinetics\Kcat) and may be estimated where the 'single-substrate' is locked in a protein cage probably  because of an active reaction site (loop 6) movement to the transition state for deprotonation; which are the on-average opened (substrate binding and release) and closed (activated for catalysis) of both monomers optimal WT (wild type) TIM conformations. Lys-12 ‹ is expected to interact with both centers, where the enediol intermediate along with the catalytic glutamate base and histidine-95 the catalytic electrophile stabalizes the reversible reaction intermediate that polarizes the substrate DHAP in the Michaelis complex. Interconversion spans the C-terminal end of the eight β-strands. For catalysis to occur likley a low pKa value transition from DHAP - for the enolase reaction enzyme enhancement 'relative to the nonenzymatic reaction - (Bound PGH - phosphoglycolohydroxamate mimics the (closed form) negative polarization (•) charge••, while PGA (2-phosphoglycolate) the positively charged residues in the two active conformation sites.) is similar for the two conformers' in the closed conformation, on ligand binding interacting with the reactive end's (β) the deprotonated substrate-bound structures to be protonated by a single-base (Glu-165) proton transfer^ mechanism.

Structure of human triose phosphate isomerase at the positions of introns in homologous TPI genes from a number of phylogenetically diverse species. The introns motif are identified as calculated in phylogeny.
Phylogenetic trees constructed on the basis of sequence comparisons for triosephosphate isomerases analysis, TIM sequences were constructed based phylogeny with similarity, to those adopting the same structural fold of interest from different species for the taxonomic groups and the K13M mutations involvement in the human triosephosphate isomerase gene family...

Interactions in the loop regions combine the effects of His95 and Lys13 for Glu165 (loop 4, 1, and 6) the three crucial catalytic residues in triose phosphate isomerase, all form the enediol intermediate necessary for the interconversion reaction catalyzed by TIM resulting in the natural substrates G3P formation. The introns motif are identified as calculated in phylogenic motifs. Poorly conserved residues as targets for specific•• drug design are expected when compared to (TPI) Triosephosphate isomerase (•). Catalytic residues of the phylogenetic relationship pathways obtained by sequence based methods of specific key amino acids can than be calculated to the incidence residues and other TIMs which may influence the (human) HsTPI equilibrium.

Non-Phosphorylating And Phosphorylating Oxidoreductase Glyceraldehyde-3-Phosphate Dehydrogenase As Part Of A Structure-Based Design In Glycolysis As The Glycolytic Protein G3PD.

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) GAPDH¹/G3PD, is located in band 12p13.31; related to both glycolysis² and gluconeogenesis-pathways. G3PD catalyzes reversible oxidative phosphorylation of inorganic phosphate and nicotinamide³ adenine dinucleotide (NAD)⁴ converting in glycolysis the glycolytic protein GAPDH⁵ in which adenosine-triphosphate (ATP)⁶ is generated when phosphoglycerate kinase (PGK)⁷ is produced in the GAPDH⁸-catalyzed reaction. These intermediate metabolites (aldolase⁹, triose-phosphate¹⁰-isomerase (TPI)¹¹) catalyze the Glycolysis reactions, in the sequence of the ten enzyme-catalyzed Embden¹²-Meyerhof¹³ reactions in the metabolic pathway. Converting phosphoglycerate mutase 1 (PGM)¹⁴ catalyzing the internal steps by 2,3-BPG¹⁵ phosphatase to form by converting D-glyceraldehyde 3-phosphate (G3P)¹⁶ into 1,3-bisphosphoglycerate (1,3-BPG)¹⁷ from its role as 3-Phosphoglyceric acid (3PG) in glycolysis as the glycolytic protein GAPDH¹⁸ that catalyzes the first step (G3P¹⁹ into 1,3-BPG) of the pathway. Plant²⁰ cells contain several reactions of photosynthesis²¹ participating in glycolysis and the Calvin-Benson²² cycle signaling pathways in plants (cytosolic-GAPC²³ (Arabidopsis thaliana)²⁴ functions in plant²⁵ cells.) its final byproduct is also another Glyceraldehyde-3-P. GAPDH is a band 3²⁶ protein that associates with the cytoplasmic²⁷ face of human erythrocyte²⁸ (RBC)²⁹ membranes. The cytoplasmic GAPDH exists primarily as a tetrameric³⁰ isoform, 4 identical 37 kDa³¹ subunits. By subcellular translocation GAPDH³² participates in nuclear events [In nuclear membrane the vesicular*³³ tubular cluster fractions³⁴ (VTCs)³⁵ - anterograde transport or retrograde³⁶ membrane transport complexes³⁷ between the intermediates, these are the Golgi³⁸ complex and the endoplasmic reticulum (ER)³⁹, in the nucleus a function is lost in disease* that exploits this process.], this a change to a non-cytosolic⁴⁰ localization due to the signal transduction pathways (considering Lm⁴¹GAPG L.⁴² mexicana⁴³-like functions.) involved in s-nitrosylase⁴⁴ activity that mediates, governed by the equilibrium between four cysteine residues (nitrosylation⁴⁵ and denitrosylation reactions)⁴⁶, inhibition of GAPDH nuclear translocation, as a basis⁴⁷ for its multifunctional⁴⁸ activities relating to the extraglycolytic functions of GAPDH. Nuclear GAPDH⁴⁹ promotes glucose metabolism to sustain⁵⁰ cellular ATP⁵¹ levels, or potentially by inhibiting targets⁵² of p300⁵³/CBP such as p53⁵⁴ dependent phosphorylation. Nitric oxide synthase or neuronal NOS ( involved in cellular and human intracellular⁵⁵ nuclei events⁵⁶, in addition to the cytoplasm) could generate nitric oxide⁵⁷ (NO). GAPDH has four cysteine⁵⁸ residues which are associated with S-nitrosylation⁵⁹-yielding NOS⁶⁰-GAPDH which “recruited” its glycolysis subunit⁶¹ from the three⁶³ molecular axes translocation roles (S-thiolation⁶⁴, S-nitrosylation or aggregated⁶⁵ enzymes (Cys-152⁶⁶ and nearby 156⁶⁷ converted into a 'cross-linked⁶⁸ soluble' states)), and (SNO⁶⁹-GAPDH) nitrosylated S-nitrosoglutathione⁷⁰ (GSNO)⁷¹ the active site cysteine residue in GAPDH at its Cys 150⁷² residue that binds to Siah1 (seven in absentia homolog 1) acquisition and the translocation of GAPDH into the nucleus, and denitrosylation using a combination of approaches, including G3P⁷³ . And NADPH may play a role in (VTC) vesicle⁷⁴ function. The complex would function in the apoptosis cascade⁷⁵ by its molecules translocation, this may⁷⁶ depend on lysine 227⁷⁷ in the human GAPDH⁷⁸-Siah⁷⁹ interaction to another intracellular position⁸⁰ induced by apoptotic⁸¹ stimuli, augments p300⁸²/CREB binding protein (CBP)-associated⁸³ acetylation of nuclear proteins. 'Engineering the cofactor (GAPDH-(Lys) K160R⁸⁴-K227A) availability prevents⁸⁵ activation of p300/CBP that interferes with GAPDH-Siah1 binding'⁸⁶-prevents the ternary (GAPDH-Siah1) complex associations translocation; that CGP-3466⁸⁷ can reduce independently with both cofactors⁸⁸. Dysregulation of protein S-nitrosylation (S-nitrosocysteine⁸⁹ - 247) by lipopolysaccharide (LPS) is associated with pathological⁹⁰ conditions which contributes to disease phenotype, where GAPDH protects ribosomal protein RP⁹¹-L13a⁹² from degradation, L13a⁹³ and GAPDH⁹⁴ forms a functional GAIT⁹⁵ complex. One of the functions of GAPDH proteins role in glycolysis⁹⁶ in relation to DNA⁹⁷ synthesis is nuclear accumulation associated by the NAD⁹⁸(+)-dependent s-nitrosylation⁹⁹ and denitrosylation⁰¹ reactions both of these isforms are distinct⁰² parallel to the uracil DNA glycosylase (UDG)⁰³ gene in mitochondria⁰⁴ and in the nucleus is N-terminally processed is the 37-kDa subunit⁰⁵ of the (GAPDH)⁰⁶ glyceraldehyde-3-phosphate dehydrogenase protein. This enzyme is an example of moonlighting protein which is validated and replaced⁰⁷ by alternative reference genes that link (in their nuclear forms) on the multifunctional⁰⁸ properties of the enzyme GAPDH⁰⁹ known as a key enzyme in glycolysis that contributes to a number of diverse cellular functions unrelated⁰⁰ to glycolysis⁰⁰¹ depending upon its subcellular location. GAPDH is a key enzyme in glycolysis the most commonly used expression is as a housekeeping⁰⁰² gene.


Cytotoxic stimuli [1a.] or Programmed cell death, via nitric oxide generation, lead to the binding of GAPDH from its usual tetrameric form to a dimeric form, to the protein Siah1 [1.] intracellular G-3-P [2.] substrate [3.] protects GAPDH from S-nitrosylation [4.]. The GAPDH-Siah interaction depends on lysine 227 [5.], in human GAPDH that interacts with a large groove [6.] of the Siah1 dimer, that connects the GAPDH dimer to PGK in the cytoplasm. The S-nitrosylation [7.,8.] abolishes catalytic activity and confers upon GAPDH the ability to bind to Siah [9.]. (GAPDH) is physiologically nitrosylated at its Cys 150 residue. GAPDH (SNO-GAPDH) [10.] binds to Siah1 [11.] by forming a protein complex. In the nucleus [12.] GAPDH is acetylated at Lys 160 [13.] and binds to the protein acetyltransferase p300/CBP. Under these conditions siah-1 formed a complex with GAPDH (PDB:4O63) and localized in the nucleus of Müller cells [14.]. GAPDH mutants [15.] that cannot bind Siah1 prevents translocation [16.] to the nucleus to elicit neurotoxicity [17.] and cell apoptosis.
[1a.] 16492755, 8769851⁰⁰³ [1.]16391220, [2.]19542219, 22534308, 3350006⁰⁰⁴, 19937139, [3.]22847419, [4.]15951807, [5.]20601085, [6.]16510976, 20392205⁰⁰⁵, [7.,8.]22817468⁰⁰⁶, 16505364⁰⁰⁷, [9.]16633896, [10.]16574384, [11.]20972425, [12.]19607794, [13.]18552833, [14.]19940145, [15.]23027902⁰⁰⁸, [16.]24362262, [17.]16492755.


Analysis of CGP-3466 Docking (NAD) to Human Placental GAPDH which decreases the synthesis of pro-apoptotic proteins is N-terminally PMID:10677844, processed to which a Rossmann NAD(P) binding fold as seen in figure 1 is a C-terminal domain as seen on this page, PMID:10617673, 26022259, 16510976 ...The structure is also used to build a model of the complex between GAPDH and the E3 ubiquitin ligase Siah1. (Purple Ribbon-1U8F_Q Figure 1.)



In the GAPDH-catalyzed reaction these intermediate metabolites (aldolase, triose-phosphate-isomerase Glycolysis and Glyconeogenesis (TPI)) catalyze the Glycolysis reactions, in the sequence of the ten enzyme-catalyzed Embden-Meyerhof reactions in the  metabolic pathway. Converting phosphoglycerate mutase 1 (PGM) catalyzing the internal steps by 2,3-BPG phosphatase to form by converting D-glyceraldehyde 3-phosphate g3p(G3P) into 1,3-bisphosphoglycerate (1,3-BPG) from its role as 3-Phosphoglyceric acid (3PG) in glycolysis as the glycolytic protein GAPDH that catalyzes the first step (G3P into 1,3-BPG) of the pathway.


GAPDH homotetramer was studied as represented an assembly of repeating spherical units that harbored a distinct birefringent crystal structure to the optic axis for the p polarization, also (r axis) discernible via transmission electron microscopy. of the relative amount of soluble monomeric GAPDH to G3P in the binding pocket of the NAD(+)-binding site residue located at the active site linked to GAPDH in Figures 5 and 6. PMID:10407144⁰⁰⁹, 25086035.


Another model building studie indicates that a structure obtained where glyceraldehyde 3-phosphate PDB:3CMC_Q binds in the P(s) pocket of the natural substrate G3P phosphorylating GAPDH (PDB:1U8F_Q) at the catalytic cysteine residue site. To define the conditions suitable for affinity for the cosubstrate, the isolation and accumulation of the intermediate metabolites per G3P monomer found in Figure 8 of the equivalent Glc-3-P structure in the binding pocket of the NAD(+)-binding site residue located at the active site linked to GAPDH. PMID:19542219, 22534308


Correctly known binding sites on ((GAPD/NAD)) structures, polar spheres of the binding catalytic pocket that corresponds to G3P (glyceraldehyde 3-phosphate) aligned to the holographical structure nonbounded spheres (salmon color), these apoenzymes together with the cofactor(s) Cys 151, 152 which corresponds as below the Ps pocket of G3P, on the Green ribbon required for cofactor activity. Together with eliminated crystallographic waters and other possible spheres, these are at least one atom of a amino acid residue in contact with at least one alpha sphere of one binding pocket on the holo protein NAD structure 1U8F_Q needed to align holo and apo structures included in this data set with G3P (PDB:3CMC_Q) was tested only on holo structure (NAD), obtained via Pea Green spheres aligned to 1U8F_Q ribbons/ligand structure which provide structural recognition insights into the biological 1U8F-Q assembly this includes 29 asymmetric units of its dimeric form, along the tetrameric 1U8F biological forms axis. PMID:9461340⁰¹⁰


(Figure 8.) These are the results without the liquid chromatography coupled mass spectrometer, that are known 3D products by two-dimensional sequence analyses with the STRAP alignment tools data sets and which may have any effect on the functions of further analysis involved in more ordered results than this study attempts to show, of the analysis that may be included are identified separated into multiple gradients here in these paired graphs. Therefore in the present work to uncover the exact coincidence of 1U8F_R and 4I7D_C, the 3D coordinates of GAPDH (PDB:1U8F_Q) to the protein Siah1 4I7D were not presenting when subjected to STRAP  alignment this apparent discrepancy (Figure 1.) was partially resolved by a (Figure 7) rendering from a more reactive native GAPDH_R homotetramer model.

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