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1.
Biochem J ; 478(13): 2425-2443, 2021 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-34198323

RESUMEN

The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologues in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified three novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.


Asunto(s)
Antivirales/química , Antivirales/farmacología , ARN Polimerasa Dependiente de ARN de Coronavirus/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Benzoatos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Chlorocebus aethiops , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Pruebas de Enzimas , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Holoenzimas/metabolismo , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Suramina/farmacología , Células Vero , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo
2.
Nat Commun ; 10(1): 1421, 2019 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-30926779

RESUMEN

Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deaza-nucleosides as potent activators (EC50 ≥ 6.5 nM) and high affinity ligands (KD ≥ 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal αD helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens.


Asunto(s)
Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Activadores de Enzimas/farmacología , Nucleósidos/análogos & derivados , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Cristalografía por Rayos X , AMP Cíclico/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/química , Dipiridamol/farmacología , Evaluación Preclínica de Medicamentos , Activadores de Enzimas/química , Holoenzimas/metabolismo , Leishmania/efectos de los fármacos , Simulación del Acoplamiento Molecular , Fosforilación/efectos de los fármacos , Transducción de Señal , Trypanosoma brucei brucei/efectos de los fármacos , Tubercidina/farmacología
3.
J Steroid Biochem Mol Biol ; 171: 80-93, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28259640

RESUMEN

Phytoestrogens are plant-derived compounds that functionally and structurally mimic mammalian estrogens. Phytoestrogens have broad inhibitory activities toward several steroidogenic enzymes, such as the 17ß-hydroxysteroid dehydrogenases (17ß-HSDs), which modulate the biological potency of androgens and estrogens in mammals. However, to date, no crystallographic data are available to explain phytoestrogens binding to mammalian 17ß-HSDs. NADP(H)-dependent 17ß-HSD from the filamentous fungus Cochliobolus lunatus (17ß-HSDcl) has been the subject of extensive biochemical, kinetic and quantitative structure-activity relationship studies that have shown that the flavonols are the most potent inhibitors. In the present study, we investigated the structure-activity relationships of the ternary complexes between the holo form of 17ß-HSDcl and the flavonols kaempferol and 3,7-dihydroxyflavone, in comparison with the isoflavones genistein and biochanin A. Crystallographic data are accompanied by kinetic analysis of the inhibition mechanisms for six flavonols (3-hydroxyflavone, 3,7-dihydroxyflavone, kaempferol, quercetin, fisetin, myricetin), one flavanone (naringenin), one flavone (luteolin), and two isoflavones (genistein, biochanin A). The kinetics analysis shows that the degree of hydroxylation of ring B significantly influences the overall inhibitory efficacy of the flavonols. A distinct binding mode defines the interactions between 17ß-HSDcl and the flavones and isoflavones. Moreover, the complex with biochanin A reveals an unusual binding mode that appears to account for its greater inhibition of 17ß-HSDcl with respect to genistein. Overall, these data provide a blueprint for identification of the distinct molecular determinants that underpin 17ß-HSD inhibition by phytoestrogens.


Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Ascomicetos/enzimología , Inhibidores Enzimáticos/metabolismo , Flavonoides/metabolismo , Proteínas Fúngicas/antagonistas & inhibidores , Modelos Moleculares , Fitoestrógenos/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/química , 17-Hidroxiesteroide Deshidrogenasas/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Bases de Datos de Proteínas , Suplementos Dietéticos , Inhibidores Enzimáticos/química , Flavonoides/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genisteína/química , Genisteína/metabolismo , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Hidroxilación , Quempferoles/química , Quempferoles/metabolismo , Conformación Molecular , Fitoestrógenos/química , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad
4.
Biochemistry ; 52(51): 9104-19, 2013 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-24279989

RESUMEN

Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O2-dependent oxidation of l-cysteine (l-Cys) to produce cysteinesulfinic acid (CSA). Adjacent to the Fe site of CDO is a covalently cross-linked cysteine-tyrosine pair (C93-Y157). While several theories have been proposed for the function of the C93-Y157 pair, the role of this post-translational modification remains unclear. In this work, the steady-state kinetics and O2/CSA coupling efficiency were measured for wild-type CDO and selected active site variants (Y157F, C93A, and H155A) to probe the influence of second-sphere enzyme-substrate interactions on catalysis. In these experiments, it was observed that both kcat and the O2/CSA coupling efficiency were highly sensitive to the presence of the C93-Y157 cross-link and its proximity to the substrate carboxylate group. Complementary electron paramagnetic resonance (EPR) experiments were performed to obtain a more detailed understanding of the second-sphere interactions identified in O2/CSA coupling experiments. Samples of the catalytically inactive substrate-bound Fe(III)-CDO species were treated with cyanide, resulting in a low-spin (S = ¹/2) ternary complex. Remarkably, both the presence of the C93-Y157 pair and interactions with the Cys carboxylate group could be readily identified by perturbations to the rhombic EPR signal. Spectroscopically validated active site quantum mechanics/molecular mechanics and density functional theory computational models are provided to suggest a potential role for Y157 in the positioning of the substrate Cys in the active site and to verify the orientation of the g-tensor relative to the CDO Fe site molecular axis.


Asunto(s)
Cisteína-Dioxigenasa/metabolismo , Hierro/metabolismo , Modelos Moleculares , Oxígeno/metabolismo , Procesamiento Proteico-Postraduccional , Sustitución de Aminoácidos , Animales , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sitios de Unión , Biocatálisis/efectos de los fármacos , Dominio Catalítico , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Cisteína-Dioxigenasa/antagonistas & inhibidores , Cisteína-Dioxigenasa/química , Cisteína-Dioxigenasa/genética , Inhibidores Enzimáticos/farmacología , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Cinética , Ratones , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxidación-Reducción/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tirosina/química , Tirosina/metabolismo
5.
Mol Cell Biochem ; 356(1-2): 83-90, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21750981

RESUMEN

Protein kinase CK2 is emerging as a target in neoplastic diseases. Inhibition of CK2 by small compounds could lead to new therapies by counteracting the elevated CK2 activities found in a variety of tumors. Currently, CK2 inhibitors are primarily evaluated by a radiometric in vitro assay tracing the amount of transferred γ-(32)P from ATP to a substrate peptide. Here, we present two alternative assays abandoning radioisotopes. The first assay is based on Förster resonance energy transfer between the fluorescence donor EDANS and the acceptor molecule DABCYL within the CK2 substrate peptide [DABCYL]-RRRDDDSDDD-[EDANS]. This peptide comprises a cleavage site for pancreatic elastase, which is located next to the phosphate acceptor serine. Only the non-phosphorylated peptide can be cleaved by elastase, disrupting the FRET effect. Thus fluorescence intensity is inversely correlated with CK2 activity. The second non-radiometric assay deploys the changing of charge that occurs within the peptide substrate RRRDDDSDDD upon phosphorylation by CK2. Substrate and product of a CK2 reaction consequently show a difference in electrophoretic mobility and thus can be separated by capillary electrophoresis. Absorption detection enabled quantification of both peptide species and allowed the determination of IC(50) values. This method facilitated the testing of a small compound library by which benzofuran derivatives were identified as potent CK2 inhibitors with IC(50) values in the submicromolar range.


Asunto(s)
Benzofuranos/química , Bioensayo/métodos , Quinasa de la Caseína II/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/análisis , Inhibidores de Proteínas Quinasas/farmacología , Radiometría/métodos , Secuencia de Aminoácidos , Animales , Quinasa de la Caseína II/metabolismo , Evaluación Preclínica de Medicamentos , Electroforesis Capilar , Transferencia Resonante de Energía de Fluorescencia , Holoenzimas/metabolismo , Humanos , Datos de Secuencia Molecular , Péptidos/química , Inhibidores de Proteínas Quinasas/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato/efectos de los fármacos , Sus scrofa
6.
Arch Microbiol ; 192(6): 437-46, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20379701

RESUMEN

Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4 degrees C with 10 microM 5'-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa +/- 5.0 kDa using Superdex 200 gel filtration. SDS-polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa +/- 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5'-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.


Asunto(s)
Euglena gracilis/enzimología , Metilmalonil-CoA Mutasa/química , Metilmalonil-CoA Mutasa/metabolismo , Propionatos/metabolismo , Secuencia de Aminoácidos , Aminoácidos/análisis , Apoenzimas/metabolismo , Cromatografía en Gel , Clonación Molecular , Cobamidas/metabolismo , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Euglena gracilis/crecimiento & desarrollo , Euglena gracilis/metabolismo , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/aislamiento & purificación , Holoenzimas/metabolismo , Cinética , Metilmalonil-CoA Mutasa/genética , Metilmalonil-CoA Mutasa/aislamiento & purificación , Mitocondrias/enzimología , Datos de Secuencia Molecular , Peso Molecular , Subunidades de Proteína/química , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Temperatura
7.
FEBS Lett ; 580(21): 5044-8, 2006 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-16949578

RESUMEN

The widely conserved SUF system is involved in Fe-S cluster repair and biogenesis. In cyanobacterium Synechocystis sp. PCC 6803, transcription of the sufBCDS operon encoding the Suf complex is negatively regulated by the upstream sufR gene encoded by the complementary strand. In this report, two promoters for the sufBCDS operon (P1 and P2) and another promoter for sufR (PsufR) was identified, and it was shown that P1 was activated by a shift to high light conditions. We also showed that Thermosynechococcus SufR negatively regulated P1 and PsufR but not P2, in a reconstituted in vitro transcription system using His(6)-tagged RNA polymerase.


Asunto(s)
Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Genes Bacterianos/genética , Proteínas Hierro-Azufre/metabolismo , Luz , Regiones Promotoras Genéticas/genética , Synechocystis/metabolismo , Transcripción Genética/efectos de la radiación , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Holoenzimas/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Operón/genética , Proteínas Recombinantes de Fusión/metabolismo , Synechocystis/genética , Synechocystis/efectos de la radiación
8.
Mol Cell Biochem ; 274(1-2): 151-61, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16335535

RESUMEN

The Drosophila clock proteins timekeeper (CK2a(Tik)) and andante (CK2beta(And)) are mutated CK2alpha and CK2beta subunits, respectively. In order to revisit the hypothesis concerning a perturbation of the beta/beta and/or alpha/beta subunit association, involving the andante mutant we have cloned, expressed and purified the recombinant andante mutant CK2beta(And) and a CK2 holoenzyme composed of CK2beta(And) and the wildtype CK2alpha subunit. Biochemical analyses using gel filtration analysis, inhibitor and heat treatment, as well as urea denaturation studies did not yield significant differences between the wildtype holoenzyme (alpha2beta2) and a holoenzyme containing wildtype CK2alpha and andante CK2beta(And). The timekeeper mutant, CK2alpha(Tik) has been reported to show a significant reduction in enzyme activity. In order to closely investigate the reason for this reduction in activity, we have also cloned and expressed the human homologue of Drosophila timekeeper. Using a CK2 holoenzyme containing the human timekeeper mutant and the wildtype CK2beta subunit we could confirm a strongly reduced activity towards CK2 substrates, but also a significant reduction in the autophosphorylation of the CK2beta in the absence of any substrate. Based on a structure-based model we postulate that the mutation M161K in Drosophila (i.e. M163K in human) is responsible for the drastic loss of activity, where the lysine residue may cause improper binding of the tri-nucleotide.


Asunto(s)
Quinasa de la Caseína II/metabolismo , Ritmo Circadiano , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Modelos Moleculares , Secuencia de Aminoácidos , Animales , Quinasa de la Caseína II/química , Quinasa de la Caseína II/genética , Proteínas de Drosophila/genética , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Hipotálamo/metabolismo , Datos de Secuencia Molecular , Mutación , Fosforilación , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido
9.
Ai Zheng ; 23(8): 874-8, 2004 Aug.
Artículo en Chino | MEDLINE | ID: mdl-15301706

RESUMEN

BACKGROUND & OBJECTIVE: Protein kinase CK2 is a ubiquitous and pleiotropic Ser/Thr protein kinase in eukaryotic cells. CK2 activity has been shown to be markedly elevated in solid tumors and leukemia cells. Its alpha or alpha' gene is a protooncogene. CK2 is attracting increasing attention as a potential target for anti-neoplastic. This study was to search specific CK2 inhibitors in tumor cells through observation of the inhibitory effects of baicalein on recombinant human protein kinase CK2 holoenzyme and its kinetics in vitro. METHODS: Recombinant human protein kinase CK2 alpha' and beta subunits were cloned and expressed by gene engineering, and purified to homogeneous. These 2 subunits were mixed at equal molar ratio to reconstitute CK2 holoenzyme. The CK2 activity was evaluated by detecting radioactivity of 32P of [gamma-32P]ATP which was incorporated into the substrate in various conditions. RESULTS: Baicalein was shown to strongly inhibit the holoenzyme activity of CK2 with IC50 of 2.54 micromol/L. Kinetic studies of baicalein on CK2 showed that baicalein acted as an inhibitor of noncompetitive with ATP(KI=7.73 micromol/L) and mixed types with casein(KI=3.07 micromol/L). CONCLUSION: Baicalein is an effective inhibitor of protein kinase CK2 in vitro.


Asunto(s)
Quinasa de la Caseína II/metabolismo , Inhibidores Enzimáticos/farmacología , Flavanonas/farmacología , Quinasa de la Caseína II/antagonistas & inhibidores , Flavanonas/aislamiento & purificación , Holoenzimas/metabolismo , Humanos , Cinética , Plantas Medicinales/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Scutellaria baicalensis/química
10.
Nature ; 423(6942): 893-7, 2003 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-12815438

RESUMEN

Escherichia coli RecBCD is a heterotrimeric helicase/nuclease that catalyses a complex reaction in which double-strand breaks in DNA are processed for repair by homologous recombination. For some time it has been clear that the RecB subunit possesses a 3' --> 5' DNA helicase activity, which was thought to drive DNA translocation and unwinding in the RecBCD holoenzyme. Here we show that purified RecD protein is also a DNA helicase, but one that possesses a 5' --> 3' polarity. We also show that the RecB and RecD helicases are both active in intact RecBCD, because the enzyme remains capable of processive DNA unwinding when either of these subunits is inactivated by mutation. These findings point to a bipolar translocation model for RecBCD in which the two DNA helicases are complementary, travelling with opposite polarities, but in the same direction, on each strand of the antiparallel DNA duplex. This bipolar motor organization helps to explain various biochemical properties of RecBCD, notably its exceptionally high speed and processivity, and offers a mechanistic insight into aspects of RecBCD function.


Asunto(s)
ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Exodesoxirribonucleasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Bases , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Exodesoxirribonucleasa V , Holoenzimas/metabolismo , Cinética , Modelos Biológicos , Plásmidos/química , Plásmidos/genética , Plásmidos/metabolismo , Subunidades de Proteína/metabolismo
11.
J Mol Biol ; 308(5): 873-82, 2001 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-11352578

RESUMEN

The genome of Dictyostelium discoideum contains a single gene (cnbA) for the regulatory (B) subunit of the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin (CN). Two mRNA species and two protein products differing in size were found. The apparent molecular masses of the protein isoforms corresponded to translation products starting from the first and second AUG codons of the primary transcript, respectively. The smaller mRNA and protein isoforms accumulated during early differentiation of the cells. Whereas the amount of the higher molecular mass protein isoform remained constant throughout development, the larger mRNA disappeared to virtually undetectable levels during aggregation. 5'RACE amplification of the smaller transcript yielded cDNAs lacking the 5' non-translated region and the first ATG initiator codon. Expression of truncated cDNAs and various chimeric genes encoding CNB-green fluorescent protein fusions in Dictyostelium indicate that the mature cnbA transcript is processed by an unconventional mechanism that leads to truncation of the 5' untranslated region and at least the first AUG initiator codon, and to utilization of the second AUG codon for translation initiation of the small CNB isoform. Determinants for this processing mechanism reside within the coding region of the cnbA gene.


Asunto(s)
Calcineurina/biosíntesis , Calcineurina/genética , Dictyostelium/genética , Procesamiento Postranscripcional del ARN , Regiones no Traducidas 5'/genética , Regiones no Traducidas 5'/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Calcineurina/química , Calcineurina/metabolismo , Calmodulina/metabolismo , Codón Iniciador/genética , Dictyostelium/citología , Dictyostelium/enzimología , Dictyostelium/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Holoenzimas/biosíntesis , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas/genética , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
12.
Biochemistry ; 39(35): 10702-10, 2000 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-10978154

RESUMEN

The crystal structures of gyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Escherichia coli have been determined in three different enzymatic states, NAD(+)-free, NAD(+)-bound, and hemiacetal intermediate. The NAD(+)-free structure reported here has been determined from monoclinic and tetragonal crystal forms. The conformational changes in GAPDH induced by cofactor binding are limited to the residues that bind the adenine moiety of NAD(+). Glyceraldehyde 3-phosphate (GAP), the substrate of GAPDH, binds to the enzyme with its C3 phosphate in a hydrophilic pocket, called the "new P(i)" site, which is different from the originally proposed binding site for inorganic phosphate. This observed location of the C3 phosphate is consistent with the flip-flop model proposed for the enzyme mechanism [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. Via incorporation of the new P(i) site in this model, it is now proposed that the C3 phosphate of GAP initially binds at the new P(i) site and then flips to the P(s) site before hydride transfer. A superposition of NAD(+)-bound and hemiacetal intermediate structures reveals an interaction between the hydroxyl oxygen at the hemiacetal C1 of GAP and the nicotinamide ring. This finding suggests that the cofactor NAD(+) may stabilize the transition state oxyanion of the hemiacetal intermediate in support of the flip-flop model for GAP binding.


Asunto(s)
Escherichia coli/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/química , NAD/química , Animales , Apoenzimas/química , Apoenzimas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Gliceraldehído 3-Fosfato/química , Gliceraldehído 3-Fosfato/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Modelos Moleculares , NAD/metabolismo , Nephropidae , Conformación Proteica , Relación Estructura-Actividad , Especificidad por Sustrato
13.
Biochem Biophys Res Commun ; 259(2): 436-42, 1999 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-10362526

RESUMEN

Methionine synthase is one of two key enzymes involved in the removal of the metabolite, homocysteine. Elevated homocysteine levels constitute a risk factor for cardiovascular diseases and for neural tube defects. In cell culture, the activity of methionine synthase is enhanced several-fold by supplementation with its cofactor, B12. The mechanism of this regulation is unknown, although it has been ascribed to a shift from apoenzyme to holoenzyme. Using sensitive assay techniques as well as a combination of Northern and Western analyses, we demonstrate that the effect of B12 on induction of methionine synthase activity is paralleled by an increase in the level of the enzyme. These studies exclude conversion of apoenzyme to holoenzyme as a basis for activation that had been described previously. Since the mRNA levels do not change during the same period that the methionine synthase levels increase, regulation of this protein by its cofactor must be exerted posttranscriptionally.


Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Vitamina B 12/farmacología , Animales , Apoenzimas/metabolismo , Sitios de Unión/genética , Línea Celular , Clonación Molecular , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Holoenzimas/metabolismo , Homocisteína/metabolismo , Humanos , Cinética , ARN Mensajero/metabolismo
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