A competent catalytic active site is necessary for substrate induced dimer assembly in triosephosphate isomerase

Jiménez‐Sandoval, Pedro; Vique‐Sánchez, José Luis; Hidalgo, Marisol López; Velázquez-Juárez, Gilberto; Díaz‐Quezada, Corina; Arroyo-Navarro, Luis Fernando; Montero-Morán, Gabriela M.; Fattori, Juliana; Díaz-Salazar, A. Jessica; Rudino‐Pinera, E.; Sotelo‐Mundo, Rogerio R.; Figueira, Ana Carolina M.; Lara-González, Samuel; Benítez‐Cardoza, Claudia G.; Brieba, Luis G.

doi:10.1016/j.bbapap.2017.07.014

Cited by 7 publications

(6 citation statements)

References 39 publications

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“…This is not a recent evolutionary event in TIM development, as studies of ancestral TIMs obtained through ancestral inference have indicated the presence of early evolutionary coupling between oligomerization and function [78]. Finally, mutagenesis studies of TIMs from the protozoan parasite Trichomonas vagina ( Tv TIM) that are capable of dissociating into stable monomers, but that dimerize once substrate binds, have shown that the dimerization is necessary for assembly of a catalytically competent active site, but that as it is being assembled, the active site itself acts to stabilize the dimer [79]. Therefore, there is clear evidence for an interplay between global conformational changes, local loop motions, and TIM function, culminating in the critical importance of ligand-gated conformational changes to drive catalysis by these enzymes.…”

Section: Loop Motion and Dimer Assembly In Triosephosphate Isomerasementioning

confidence: 99%

The role of ligand-gated conformational changes in enzyme catalysis

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Calixto

Richard

et al. 2019

Biochemical Society Transactions

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Structural and biochemical studies on diverse enzymes have highlighted the importance of ligand-gated conformational changes in enzyme catalysis, where the intrinsic binding energy of the common phosphoryl group of their substrates is used to drive energetically unfavorable conformational changes in catalytic loops, from inactive open to catalytically competent closed conformations. However, computational studies have historically been unable to capture the activating role of these conformational changes. Here, we discuss recent experimental and computational studies, which can remarkably pinpoint the role of ligand-gated conformational changes in enzyme catalysis, even when not modeling the loop dynamics explicitly. Finally, through our joint analyses of these data, we demonstrate how the synergy between theory and experiment is crucial for furthering our understanding of enzyme catalysis.

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Section: Loop Motion and Dimer Assembly In Triosephosphate Isomerasementioning

confidence: 99%

The role of ligand-gated conformational changes in enzyme catalysis

Moreira

Calixto

Richard

et al. 2019

Biochemical Society Transactions

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“…In all TPIs studied to date, this enzyme is an obligatory dimer, meaning that TPI monomers are catalytically incompetent (Zarate‐Perez et al ., ; Jiménez‐Sandoval et al ., ). To assess the role of monomerization in catalysis, we measured the specific activities of wild‐type and point mutant AtcTPIs incubating the enzymes for 24 h at 25°C at protein concentrations that rank from 50 ng mL −1 to 1000 μg mL −1 (Table ).…”

Section: Resultsmentioning

confidence: 97%

“…The interaction between loop 1 from one subunit and loop 3 from the neighbor subunit is critical for dimer formation. For instance, engineered monomeric versions of several TPIs were designed by the decreasing in the length of loop 3 (Borchert et al ., ; Saab‐Rincon et al ., ; Jiménez‐Sandoval et al ., ). Derivatization of the residue to corresponding AtcTPI‐Cys13 with alkylating agents generate TPIs monomers in TPIs from P. falciparum and E. histolytica , and also protein aggregation in TPIs from T. cruzi and T. brucei (Garza‐Ramos et al ., ; Maithal et al ., ; Rodríguez‐Romero et al ., ).…”

Section: Resultsmentioning

confidence: 97%

“…Triosephosphate isomerase (TPI) interconverts glyceraldehyde 3‐phosphate to dihydroxyacetone near the rate diffusion limit during glycolysis, gluconeogenesis, and the Calvin−Benson cycle (Blacklow et al ., ; Wierenga et al ., ; Michelet et al ., ). TPI folds as a (β−α) 8 barrel and its dimerization is necessary to assemble a functional active site (Banner et al ., ; Alber et al ., ; Wierenga, ; Lara‐Gonzalez et al ., ; Jiménez‐Sandoval et al ., ). Dimerization is achieved by an extensive set of protein−protein interactions, and among these, an interaction between loop1 of one subunit with loop 3 from the neighboring subunit is the main contributor to the dimer interface area (Wierenga et al ., ; Lara‐Gonzalez et al ., ).…”

Section: Introductionmentioning

confidence: 97%

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Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox‐based modifications

et al. 2019

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Summary Reactive oxidative species (ROS) and S‐glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI‐Cys13 and At‐Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI‐Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI‐Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI‐Cys13, mutations in AtcTPI‐Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI‐Cys13 and AtcTPI‐Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI‐Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent‐exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S‐glutathionylation protects AtcTPI from irreversible chemical modifications and re‐routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.

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“…Stock solutions of EQX-NO derivatives were prepared in DMSO (1 %) and further diluted with media to obtain 50 and 100 µg µL -1 of each compound tested. Recombinant TvTIM was obtained as previously described (27,28). The conversion of glyceraldehyde 3-phosphate (GAP) into dihydroxyacetone phosphate was followed to determine enzyme activity following a previous procedure (29)(30)(31)(32)(33)(34)(35).…”

Section: In Vitro Assays Against Tvtimmentioning

confidence: 99%

Esters of quinoxaline-7-carboxylate-1,4-di-N-oxide as Trichomonas vaginalis triosephosphate isomerase inhibitors

et al. 2020

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Trichomoniasis is a public health problem worldwide. Trichomoniasis treatment consists of the use of nitroimidazole derivatives; however, therapeutic ineffectiveness occurs in 5 to 20 % of the cases. Therefore, it is essential to propose new pharmacological agents against this disease. In this work, esters of quinoxaline-7-carboxylate-1,4-di-N-oxide (EQX-NO) were evaluated in in vitro assays as novel trichomonicidal agents. Additionally, an in vitro enzyme assay and molecular docking analysis against triosephosphate isomerase of Trichomonas vaginalis to confirm their mechanism of action were performed. Ethyl (compound 12) and n-propyl (compound 37) esters of quinoxaline-7-carboxy-late-1,4-di-N-oxide derivatives showed trichomonicidal activity comparable to nitazoxanide, whereas five methyl (compounds 5, 15, 19, 20 and 22), four isopropyl (compounds 28, 29, 30 and 34), three ethyl (compounds 4, 13 and 23) and one npropyl (compound 35) ester derivatives displayed activity comparable to albendazole. Compounds 6 and 20 decreased 100 % of the enzyme activity of recombinant protein triosephosphate isomerase.

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A competent catalytic active site is necessary for substrate induced dimer assembly in triosephosphate isomerase

Cited by 7 publications

References 39 publications

The role of ligand-gated conformational changes in enzyme catalysis

The role of ligand-gated conformational changes in enzyme catalysis

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox‐based modifications

Esters of quinoxaline-7-carboxylate-1,4-di-N-oxide as Trichomonas vaginalis triosephosphate isomerase inhibitors

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