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1.
Proteins ; 85(7): 1190-1211, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28378917

RESUMEN

Triosephosphate isomerase (TIM) is a ubiquitous enzyme, which appeared early in evolution. TIM is responsible for obtaining net ATP from glycolysis and producing an extra pyruvate molecule for each glucose molecule, under aerobic and anaerobic conditions. It is placed in a metabolic crossroad that allows a quick balance of the triose phosphate aldolase produced by glycolysis, and is also linked to lipid metabolism through the alternation of glycerol-3-phosphate and the pentose cycle. TIM is one of the most studied enzymes with more than 199 structures deposited in the PDB. The interest for this enzyme stems from the fact that it is involved in glycolysis, but also in aging, human diseases and metabolism. TIM has been a target in the search for chemical compounds against infectious diseases and is a model to study catalytic features. Until February 2017, 62% of all residues of the protein have been studied by mutagenesis and/or using other approaches. Here, we present a detailed and comprehensive recompilation of the reported effects on TIM catalysis, stability, druggability and human disease produced by each of the amino acids studied, contributing to a better understanding of the properties of this fundamental protein. The information reviewed here shows that the role of the noncatalytic residues depend on their molecular context, the delicate balance between the short and long-range interactions in concerted action determining the properties of the protein. Each protein should be regarded as a unique entity that has evolved to be functional in the organism to which it belongs. Proteins 2017; 85:1190-1211. © 2017 Wiley Periodicals, Inc.


Asunto(s)
Inhibidores Enzimáticos/química , Triosa-Fosfato Isomerasa/química , Secuencia de Aminoácidos , Biocatálisis , Dominio Catalítico , Estabilidad de Enzimas , Humanos , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por Sustrato , Triosa-Fosfato Isomerasa/antagonistas & inhibidores , Triosa-Fosfato Isomerasa/genética , Triosa-Fosfato Isomerasa/metabolismo
2.
Molecules ; 20(8): 14595-610, 2015 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-26274947

RESUMEN

The current pharmacological Chagas disease treatments, using Nifurtimox or Benznidazole, show limited therapeutic results and are associated with potential side effects, like mutagenicity. Using random screening we have identified new chemotypes that were able to inhibit relevant targets of the Trypanosoma cruzi. We found 3H-[1,2]dithioles with the ability to inhibit Trypanosoma cruzi triosephosphate isomerase (TcTIM). Herein, we studied the structural modifications of this chemotype to analyze the influence of volume, lipophilicity and electronic properties in the anti-T. cruzi activity. Their selectivity to parasites vs. mammalian cells was also examined. To get insights into a possible mechanism of action, the inhibition of the enzymatic activity of TcTIM and cruzipain, using the isolated enzymes, and the inhibition of membrane sterol biosynthesis and excreted metabolites, using the whole parasite, were achieved. We found that this structural framework is interesting for the generation of innovative drugs for the treatment of Chagas disease.


Asunto(s)
Tolueno/análogos & derivados , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Macrófagos/efectos de los fármacos , Ratones , Esteroles/antagonistas & inhibidores , Esteroles/biosíntesis , Tolueno/síntesis química , Tolueno/química , Tolueno/farmacología , Tripanocidas/síntesis química , Trypanosoma cruzi/metabolismo
3.
Proteins ; 82(2): 323-35, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23966267

RESUMEN

It is generally assumed that the amino acids that exist in all homologous enzymes correspond to residues that participate in catalysis, or that are essential for folding and stability. Although this holds for catalytic residues, the function of conserved noncatalytic residues is not clear. It is not known if such residues are of equal importance and have the same role in different homologous enzymes. In humans, the E104D mutation in triosephosphate isomerase (TIM) is the most frequent mutation in the autosomal diseases named "TPI deficiencies." We explored if the E104D mutation has the same impact in TIMs from four different organisms (Homo sapiens, Giardia lamblia, Trypanosoma cruzi, and T. brucei). The catalytic properties were not significantly affected by the mutation, but it affected the rate and extent of formation of active dimers from unfolded monomers differently. Scanning calorimetry experiments indicated that the mutation was in all cases destabilizing, but the mutation effect on rates of irreversible denaturation and transition-state energetics were drastically dependent on the TIM background. For instance, the E104D mutation produce changes in activation energy ranging from 430 kJ mol(-1) in HsTIM to -78 kJ mol(-1) in TcTIM. Thus, in TIM the role of a conserved noncatalytic residue is drastically dependent on its molecular background. Accordingly, it would seem that because each protein has a particular sequence, and a distinctive set of amino acid interactions, it should be regarded as a unique entity that has evolved for function and stability in the organisms to which it belongs.


Asunto(s)
Proteínas Protozoarias/química , Triosa-Fosfato Isomerasa/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia Conservada , Cristalografía por Rayos X , Entropía , Estabilidad de Enzimas , Giardia lamblia/enzimología , Humanos , Cinética , Modelos Moleculares , Desplegamiento Proteico , Proteínas Protozoarias/genética , Homología Estructural de Proteína , Triosa-Fosfato Isomerasa/genética , Trypanosoma brucei brucei/enzimología , Trypanosoma cruzi/enzimología
4.
J Enzyme Inhib Med Chem ; 29(2): 198-204, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23406473

RESUMEN

CONTEXT: Triosephosphate isomerase (TIM) is a ubiquitous enzyme that has been targeted for the discovery of new small molecular weight compounds used against Trypanosoma cruzi, the causative agent of Chagas disease. We have identified phenazine and 1,2,6-thiadiazine chemotypes as novel inhibitors of TIM from T. cruzi (TcTIM). OBJECTIVE: Study the mechanism of TcTIM inhibition by a phenazine derivative and by a 1,2,6-thiadiazine derivative. METHODS: We performed biochemical and theoretical molecular docking studies to characterize the interaction of the derivatives with wild-type and mutant TcTIM. RESULTS AND CONCLUSION: At low micromolar concentrations, the compounds induce highly selective irreversible inactivation of parasitic TIM. The molecular docking simulations indicate that the phenazine derivative likely interferes with the association of the two monomers of the dimeric enzyme by locating at the dimer interface, while 1,2,6-thiadiazine could act as an inhibitor binding to a region surrounding Cys-118.


Asunto(s)
Antiprotozoarios/farmacología , Inhibidores Enzimáticos/farmacología , Fenazinas/farmacología , Tiadiazinas/farmacología , Triosa-Fosfato Isomerasa/antagonistas & inhibidores , Trypanosoma cruzi/efectos de los fármacos , Antiprotozoarios/química , Unión Competitiva , Enfermedad de Chagas/tratamiento farmacológico , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/química , Escherichia coli/genética , Modelos Biológicos , Simulación del Acoplamiento Molecular , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Fenazinas/química , Unión Proteica , Pliegue de Proteína , Multimerización de Proteína , Tiadiazinas/química , Triosa-Fosfato Isomerasa/química , Triosa-Fosfato Isomerasa/genética , Trypanosoma cruzi/enzimología
5.
Parasitology ; 139(13): 1729-38, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22931930

RESUMEN

The glycolytic enzyme triosephosphate isomerase catalyses the isomerization between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Here we report that Trichomonas vaginalis contains 2 fully functional tpi genes. Both genes are located in separated chromosomal context with different promoter regulatory elements and encode ORFs of 254 amino acids; the only differences between them are the character of 4 amino acids located in α-helices 1, 2 and 8. Semi-quantitative RT-PCR assays showed that tpi2 transcript is approximately 3·3-fold more abundant than tpi1. Using an anti-TvTIM2 polyclonal antibody it was demonstrated that TIM proteins have a cytoplasmic localization and both enzymes are able to complement an Escherichia coli strain carrying a deletion of its endogenous tpi gene. Both TIM proteins assemble as dimers and their secondary structure assessment is essentially identical to TIM from Saccharomyces cerevisiae. The kinetic catalytic constants of the recombinant enzymes using glyceraldehyde-3-phosphate as substrate are similar to the catalytic constants of TIMs from other organisms including parasitic protozoa. As T. vaginalis depends on glycolysis for ATP production, we speculate 2 possible reasons to maintain a duplicated tpi copy on its genome: an increase in gene dosage or an early event of neofunctionalization of TIM as a moonlighting protein.


Asunto(s)
Trichomonas vaginalis/enzimología , Trichomonas vaginalis/genética , Triosa-Fosfato Isomerasa/genética , Triosa-Fosfato Isomerasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Citoplasma/enzimología , Escherichia coli/genética , Duplicación de Gen , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Prueba de Complementación Genética , Modelos Moleculares , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Triosa-Fosfato Isomerasa/química
6.
J Bioenerg Biomembr ; 40(6): 561-8, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19139978

RESUMEN

Although the capacity of isolated beta-subunits of the ATP synthase/ATPase to perform catalysis has been extensively studied, the results have not conclusively shown that the subunits are catalytically active. Since soluble F(1) of mitochondrial H(+)-ATPase can bind inorganic pyrophosphate (PP(i)) and synthesize PP(i) from medium phosphate, we examined if purified His-tagged beta-subunits from Thermophilic bacillus PS3 can hydrolyze PP(i). The difference spectra in the near UV CD of beta-subunits with and without PP(i) show that PP(i) binds to the subunits. Other studies show that beta-subunits hydrolyze [(32)P] PP(i) through a Mg(2+)-dependent process with an optimal pH of 8.3. Free Mg(2+) is required for maximal hydrolytic rates. The Km for PP(i) is 75 microM and the Vmax is 800 pmol/min/mg. ATP is a weak inhibitor of the reaction, it diminishes the Vmax and increases the Km for PP(i). Thus, isolated beta-subunits are catalytically competent with PP(i) as substrate; apparently, the assembly of beta-subunits into the ATPase complex changes substrate specificity, and leads to an increase in catalytic rates.


Asunto(s)
Complejos de ATP Sintetasa/química , Complejos de ATP Sintetasa/metabolismo , Archaea/enzimología , Proteínas Bacterianas/química , Complejos de ATP Sintetasa/aislamiento & purificación , Catálisis , Activación Enzimática , Estabilidad de Enzimas , Hidrólisis , Solubilidad
7.
Artículo en Inglés | MEDLINE | ID: mdl-18678934

RESUMEN

The PyrR transcriptional regulator is widely distributed in bacteria. This RNA-binding protein is involved in the control of genes involved in pyrimidine biosynthesis, in which uridyl and guanyl nucleotides function as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of two crystal forms of Bacillus halodurans PyrR are reported. One of the forms belongs to the monoclinic space group P2(1) with unit-cell parameters a = 59.7, b = 87.4, c = 72.1 A, beta = 104.4 degrees , while the other form belongs to the orthorhombic space group P22(1)2(1) with unit-cell parameters a = 72.7, b = 95.9, c = 177.1 A. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of four and six monomers per asymmetric unit; a crystallographic tetramer is formed in both forms.


Asunto(s)
Bacillus/química , Proteínas Bacterianas/química , Pentosiltransferasa/química , Proteínas Represoras/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Cromatografía en Gel , Clonación Molecular , Cristalografía por Rayos X , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Modelos Moleculares , Pentosiltransferasa/genética , Pentosiltransferasa/aislamiento & purificación , Conformación Proteica , Proteínas Represoras/genética , Proteínas Represoras/aislamiento & purificación
8.
Proteins ; 67(1): 75-83, 2007 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-17221869

RESUMEN

Homodimeric triosephosphate isomerase (TIM) from Trypanosoma cruzi (TcTIM) and T. brucei (TbTIM) are markedly similar in amino acid sequence and three-dimensional structure. In their dimer interfaces, each monomer has a Cys15 that is surrounded by loop3 of the adjoining subunit. Perturbation of Cys15 by methylmethane thiosulfonate (MMTS) induces abolition of catalysis and structural changes. In the two TIMs, the structural arrangements of their Cys15 are almost identical. Nevertheless, the susceptibility of TcTIM to MMTS is nearly 100-fold higher than in TbTIM. To ascertain the extent to which the characteristics of the interface Cys depend on the dynamics of its own monomer or on those of the adjacent monomer, we studied MMTS action on mutants of TcTIM that had the interface residues of TbTIM, and hybrids that have only one interfacial Cys15 (C15ATcTIM-wild type TbTIM). We found that the solvent exposure of the interfacial Cys depends predominantly on the characteristics of the adjoining monomer. The maximal inhibition of activity induced by perturbation of the sole interface Cys in the C15ATcTIM-TbTIM hybrid is around 60%. Hybrids formed with C15ATcTIM monomers and catalytically inert TbTIM monomers (E168DTbTIM) were also studied. Their activity drops by nearly 50% when the only interfacial Cys is perturbed. These results in conjunction with those on C15ATcTIM-wild type TbTIM hybrid indicate that about half of the activity of each monomer depends on the integrity of each of the two Cys15-loop3 portions of the interface. This could be another reason of why TIM is an obligatory dimer.


Asunto(s)
Cisteína/química , Metilmetanosulfonato/análogos & derivados , Triosa-Fosfato Isomerasa/química , Trypanosoma brucei brucei/enzimología , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Dimerización , Cinética , Metilmetanosulfonato/química , Metilmetanosulfonato/farmacología , Mutagénesis Sitio-Dirigida , Mapeo de Interacción de Proteínas/métodos , Estructura Cuaternaria de Proteína , Triosa-Fosfato Isomerasa/antagonistas & inhibidores , Triosa-Fosfato Isomerasa/genética , Triosa-Fosfato Isomerasa/metabolismo
9.
Biotechnol Rep (Amst) ; 13: 42-48, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28352562

RESUMEN

Rare arginine codons AGA and AGG affect the heterologous expression of proteins in Eschericha coli. The tRNAs necessary for protein synthesis are scarce in E. coli strain BL21(DE3) pLysS and plentiful in strain BL21(DE3) CodonPlus -RIL. We evaluated in both bacterial strains the effect of these rare codons on the expression of triosephosphate isomerases from 7 different species, whose sequences had different dispositions of rare arginine codons. The ratio of expressed protein (CP/Bl21) correlated with the number of rare codons. Our study shows that the number, position and particularities of the combination of rare Arg codons in the natural non-optimized sequences of the triosephosphate isomerases influence the synthesis of heterologous proteins in E. coli and could have implications in the selection of better sequences for engineering enzymes for novel or manipulated metabolic pathways or for the expression levels of non enzymatic proteins..

10.
ChemMedChem ; 11(12): 1328-38, 2016 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-26492824

RESUMEN

Triosephosphate isomerase (TIM) is an essential Trypanosoma cruzi enzyme and one of the few validated drug targets for Chagas disease. The known inhibitors of this enzyme behave poorly or have low activity in the parasite. In this work, we used symmetrical diarylideneketones derived from structures with trypanosomicidal activity. We obtained an enzymatic inhibitor with an IC50 value of 86 nm without inhibition effects on the mammalian enzyme. These molecules also affected cruzipain, another essential proteolytic enzyme of the parasite. This dual activity is important to avoid resistance problems. The compounds were studied in vitro against the epimastigote form of the parasite, and nonspecific toxicity to mammalian cells was also evaluated. As a proof of concept, three of the best derivatives were also assayed in vivo. Some of these derivatives showed higher in vitro trypanosomicidal activity than the reference drugs and were effective in protecting infected mice. In addition, these molecules could be obtained by a simple and economic green synthetic route, which is an important feature in the research and development of future drugs for neglected diseases.


Asunto(s)
Antiprotozoarios/farmacología , Cisteína Endopeptidasas/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Triosa-Fosfato Isomerasa/antagonistas & inhibidores , Trypanosoma cruzi/efectos de los fármacos , Animales , Antiprotozoarios/química , Antiprotozoarios/uso terapéutico , Sitios de Unión , Enfermedad de Chagas/tratamiento farmacológico , Cisteína Endopeptidasas/química , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Cetonas/química , Cetonas/farmacología , Cetonas/uso terapéutico , Ratones , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , Triosa-Fosfato Isomerasa/metabolismo , Trypanosoma cruzi/crecimiento & desarrollo
11.
J Mol Biol ; 341(5): 1355-65, 2004 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-15321726

RESUMEN

We characterized by crystallographic, calorimetric and biochemical methods the action of a low molecular weight compound, 3-(2-benzothiazolylthio)-1-propanesulfonic acid (compound 8) that binds to the dimer interface of triosephosphate isomerase from Trypanosoma cruzi (TcTIM) and thereby abolishes its function with a high level of selectivity. The kinetics of TcTIM inactivation by the agent and isothermal titration calorimetry experiments showed that the binding of two molecules of the compound per enzyme is needed for inactivation. The binding of the first molecule is endothermic, and that of the second exothermic. Crystals of TcTIM in complex with one molecule of the inactivating agent that diffracted to a resolution of 2A were obtained. The compound is at the dimer interface at less than 4A from residues of the two subunits. Compound 8 is more effective at low than at high protein concentrations, indicating that it perturbs the association between the two TcTIM monomers. Calorimetric and kinetic data of experiments in which TcTIM was added to a solution of the inactivating agent showed that at low concentrations of the compound, inactivation is limited by binding, whereas at high concentrations of the agent, the events that follow binding become rate-limiting. The portion of the interface of TcTIM that binds the benzothiazole derivative and its equivalent region in human TIM differs in amino acid composition and hydrophobic packing. Thus, we show that by focusing on protein-protein interfaces, it is possible to discover low molecular weight compounds that are selective for enzymes from parasites.


Asunto(s)
Ácidos Alcanesulfónicos/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Triosa-Fosfato Isomerasa/química , Triosa-Fosfato Isomerasa/metabolismo , Trypanosoma cruzi/enzimología , Ácidos Alcanesulfónicos/química , Animales , Sitios de Unión , Calorimetría , Dimerización , Humanos , Modelos Moleculares , Estructura Molecular , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo
12.
PLoS One ; 10(11): e0141747, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26618356

RESUMEN

The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.


Asunto(s)
Multimerización de Proteína , Proteínas Protozoarias/química , Trichomonas vaginalis/enzimología , Triosa-Fosfato Isomerasa/química , Secuencia de Aminoácidos , Dominio Catalítico , Estabilidad de Enzimas , Datos de Secuencia Molecular , Unión Proteica , Proteínas Protozoarias/metabolismo , Triosa-Fosfato Isomerasa/metabolismo
13.
Eur J Med Chem ; 100: 246-56, 2015 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-26094151

RESUMEN

The neglected disease American trypanosomiasis is one of the major health problems in Latin America. Triosephosphate isomerase from Trypanosoma cruzi (TcTIM), the etiologic agent of this disease, has been proposed as a druggable target. Some bis-benzothiazoles have been described as irreversible inhibitors of this enzyme. On the other hand, new bioactive furane-containing thiazoles have been described as excellent in vivo anti-T. cruzi agents. This encouraged us to design and develop new bis-thiazoles with potential use as drugs for American trypanosomiasis. The bis-thiazol 5, 3,3'-allyl-2,2'-bis[3-(2-furyl)-2-propenylidenehydrazono]-2,2',3,3'-tetrahydro-4,4'-bisthiazole, showed the best in vitro anti-T. cruzi profile with a higher selectivity index than the reference drugs Nifurtimox and Benznidazole against amastigote form of the parasite. This derivative displayed marginal activity against TcTIM however the bis-thiazol 14, 3-allyl-2-[3-(2-furyl)-2-propenylidenehydrazono]-3'-phenyl-2'-(3-phenyl-2-propenylidenehydrazono]-2,2',3,3'-tetrahydro-4,4'-bisthiazole, was an excellent inhibitor of the enzyme of the parasite. The absence of both in vitro mutagenic and in vivo toxicity effects, together with the activity of bis-thiazol 5in vivo, suggests that this compound is a promising anti-T. cruzi agent surpassing the "hit-to-lead" stage in the drug development process.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Tiazoles/farmacología , Triosa-Fosfato Isomerasa/antagonistas & inhibidores , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Interacciones Hidrofóbicas e Hidrofílicas , Macrófagos , Ratones , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/química , Triosa-Fosfato Isomerasa/metabolismo , Tripanocidas/síntesis química , Tripanocidas/química
14.
Proteins ; 48(3): 580-90, 2002 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-12112681

RESUMEN

The susceptibility to subtilisin of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) was studied. Their amino sequence and 3D structure are markedly similar. In 36 h of incubation at a molar ratio of 4 TIM per subtilisin, TcTIM underwent extensive hydrolysis, loss of activity, and large structural alterations. Under the same conditions, only about 50% of the monomers of TbTIM were cleaved in two sites. The higher sensitivity of TcTIM to subtilisin is probably due to a higher intrinsic flexibility. We isolated and characterized TbTIM that had been exposed to subtilisin. It exhibited the molecular mass of the dimer, albeit it was formed by one intact and one nicked monomer. Its k(cat) with glyceraldehyde 3-phosphate was half that of native TbTIM, with no change in K(m). The intrinsic fluorescence of nicked TbTIM was red-shifted by 5 nm. The association between subunits was not affected. The TbTIM data suggest that there are structural differences in the two monomers or that alterations of one subunit change the characteristics of the other subunit. In comparison to the action of subtilisin on TIMs from other species, the trypanosomal enzymes appear to be unique.


Asunto(s)
Triosa-Fosfato Isomerasa/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Electroforesis , Estabilidad de Enzimas , Hidrólisis , Cinética , Datos de Secuencia Molecular , Peso Molecular , Alineación de Secuencia , Subtilisina/metabolismo , Triosa-Fosfato Isomerasa/química , Trypanosoma brucei brucei/patogenicidad , Trypanosoma cruzi/patogenicidad
15.
Curr Top Med Chem ; 2(5): 457-70, 2002 May.
Artículo en Inglés | MEDLINE | ID: mdl-11966467

RESUMEN

Millions of people worldwide are infected by some kind of parasite and millions are in risk of contracting infection. In addition, it is now accepted that parasites are rapidly developing resistance to drugs that a few years ago were effective. This gloom picture underscores the urgent need to develop new drugs against parasitic diseases. Fortunately, the important technological advances that have been made in the past years will, in principle, facilitate the discovery of new and effective agents against parasitic diseases. In many of the approaches for drug design the basic premise is the identification of a macromolecule that is central to the life of the parasite. Because the life of all living organisms depends on multiple protein-protein interactions and the function of oligomeric proteins, it is worthwhile to explore if protein interfaces could be exploited for drug design. Here we review some of the work that has been done in this direction, and attempt to call attention to the richness of protein-protein interfaces for the design of agents that could lead to the development of drugs against parasitic diseases.


Asunto(s)
Inhibidores Enzimáticos/química , Parásitos/efectos de los fármacos , Animales , Dimerización , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Parásitos/enzimología , Unión Proteica/efectos de los fármacos
16.
PLoS One ; 6(4): e18791, 2011 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-21533154

RESUMEN

For a better comprehension of the structure-function relationship in proteins it is necessary to identify the amino acids that are relevant for measurable protein functions. Because of the numerous contacts that amino acids establish within proteins and the cooperative nature of their interactions, it is difficult to achieve this goal. Thus, the study of protein-ligand interactions is usually focused on local environmental structural differences. Here, using a pair of triosephosphate isomerase enzymes with extremely high homology from two different organisms, we demonstrate that the control of a seventy-fold difference in reactivity of the interface cysteine is located in several amino acids from two structurally unrelated regions that do not contact the cysteine sensitive to the sulfhydryl reagent methylmethane sulfonate, nor the residues in its immediate vicinity. The change in reactivity is due to an increase in the apparent pKa of the interface cysteine produced by the mutated residues. Our work, which involved grafting systematically portions of one protein into the other protein, revealed unsuspected and multisite long-range interactions that modulate the properties of the interface cysteines and has general implications for future studies on protein structure-function relationships.


Asunto(s)
Aminoácidos/química , Triosa-Fosfato Isomerasa/metabolismo , Trypanosoma/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biocatálisis , Cartilla de ADN , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Triosa-Fosfato Isomerasa/química , Triosa-Fosfato Isomerasa/genética
17.
Insect Biochem Mol Biol ; 41(6): 400-9, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21396445

RESUMEN

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 µmol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.


Asunto(s)
Embrión no Mamífero/enzimología , Proteínas Recombinantes/metabolismo , Rhipicephalus/enzimología , Triosa-Fosfato Isomerasa/metabolismo , Cigoto/enzimología , Secuencia de Aminoácidos , Animales , Catálisis , Clonación Molecular , Cristalografía por Rayos X , Cisteína/química , Cisteína/metabolismo , Dihidroxiacetona Fosfato/metabolismo , Dimerización , Escherichia coli , Gliceraldehído 3-Fosfato/metabolismo , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica/efectos de los fármacos , Proteínas Recombinantes/genética , Rhipicephalus/embriología , Alineación de Secuencia , Reactivos de Sulfhidrilo/farmacología , Triosa-Fosfato Isomerasa/antagonistas & inhibidores , Triosa-Fosfato Isomerasa/genética , Triosa-Fosfato Isomerasa/aislamiento & purificación
18.
PLoS One ; 6(6): e21035, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21738601

RESUMEN

We previously observed that human homodimeric triosephosphate isomerase (HsTIM) expressed in Escherichia coli and purified to apparent homogeneity exhibits two significantly different thermal transitions. A detailed exploration of the phenomenon showed that the preparations contain two proteins; one has the expected theoretical mass, while the mass of the other is 28 Da lower. The two proteins were separated by size exclusion chromatography in 3 M urea. Both proteins correspond to HsTIM as shown by Tandem Mass Spectrometry (LC/ESI-MS/MS). The two proteins were present in nearly equimolar amounts under certain growth conditions. They were catalytically active, but differed in molecular mass, thermostability, susceptibility to urea and proteinase K. An analysis of the nucleotides in the human TIM gene revealed the presence of six codons that are not commonly used in E. coli. We examined if they were related to the formation of the two proteins. We found that expression of the enzyme in a strain that contains extra copies of genes that encode for tRNAs that frequently limit translation of heterologous proteins (Arg, Ile, Leu), as well as silent mutations of two consecutive rare Arg codons (positions 98 and 99), led to the exclusive production of the more stable protein. Further analysis by LC/ESI-MS/MS showed that the 28 Da mass difference is due to the substitution of a Lys for an Arg residue at position 99. Overall, our work shows that two proteins with different biochemical and biophysical properties that coexist in the same cell environment are translated from the same nucleotide sequence frame.


Asunto(s)
Arginina/genética , Escherichia coli/metabolismo , Lisina/genética , Triosa-Fosfato Isomerasa/química , Triosa-Fosfato Isomerasa/metabolismo , Arginina/química , Rastreo Diferencial de Calorimetría , Cromatografía en Gel , Cromatografía Liquida , Biología Computacional , Escherichia coli/genética , Humanos , Lisina/química , Polimorfismo Genético/genética , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Triosa-Fosfato Isomerasa/genética
19.
Eur J Med Chem ; 45(12): 5767-72, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20889239

RESUMEN

Triosephosphate isomerase from Trypanosoma cruzi (TcTIM), an enzyme in the glycolytic pathway that exhibits high catalytic rates of glyceraldehyde-3-phosphate- and dihydroxyacetone-phosphate-isomerization only in its dimeric form, was screened against an in-house chemical library containing nearly 230 compounds belonging to different chemotypes. After secondary screening, twenty-six compounds from eight different chemotypes were identified as screening positives. Four compounds displayed selectivity for TcTIM over TIM from Homo sapiens and, concomitantly, in vitro activity against T. cruzi.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Triosa-Fosfato Isomerasa/antagonistas & inhibidores , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Dimerización , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Modelos Moleculares , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Estereoisomerismo , Relación Estructura-Actividad , Tripanocidas/síntesis química , Tripanocidas/química , Trypanosoma cruzi/enzimología
20.
J Mol Biol ; 385(3): 924-37, 2009 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-18992756

RESUMEN

Theoretical, computational, and experimental studies have suggested the existence of solvation barriers in protein unfolding and denaturation processes. These barriers are related to the finite size of water molecules and can be envisioned as arising from the asynchrony between water penetration and breakup of internal interactions. Solvation barriers have been proposed to play roles in protein cooperativity and kinetic stability; therefore, they may be expected to be subject to natural selection. We study the thermal denaturation, in the presence and in the absence of chemical denaturants, of triosephosphate isomerases (TIMs) from three different species: Trypanosoma cruzi, Trypanosoma brucei, and Leishmania mexicana. In all cases, denaturation was irreversible and kinetically controlled. Surprisingly, however, we found large differences between the kinetic denaturation parameters, with T. cruzi TIM showing a much larger activation energy value (and, consequently, much lower room-temperature, extrapolated denaturation rates). This disparity cannot be accounted for by variations in the degree of exposure to solvent in transition states (as measured by kinetic urea m values) and is, therefore, to be attributed mainly to differences in solvation-barrier contributions. This was supported by structure-energetics analyses of the transition states and by application of a novel procedure to estimate from experimental data the solvation-barrier impact at the entropy and free-energy levels. These analyses were actually performed with an extended protein set (including six small proteins plus seven variants of lipase from Thermomyces lanuginosus and spanning a wide range of activation parameters), allowing us to delineate the general trends of the solvation-barrier contributions. Overall, this work supports that proteins sharing the same structure and function but belonging to different organisms may show widely different solvation barriers, possibly as a result of different levels of the selection pressure associated with cooperativity, kinetic stability, and related factors.


Asunto(s)
Trypanosoma/metabolismo , Animales , Rastreo Diferencial de Calorimetría , Cinética , Modelos Teóricos , Desnaturalización Proteica , Solubilidad , Especificidad de la Especie , Termodinámica
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