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1.
Nat Prod Res ; 36(12): 3153-3157, 2022 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-34219561

RESUMEN

Arginine kinase from Trypanosoma cruzi (TcAK) catalyzes the interconversion of arginine and phosphoarginine to maintain the ATP/ADP cell balance, and is involved in the parasites' energetic homeostasis and stress responses. Using virtual screening approaches, some plant-derived polyphenolic pigments, such as anthocyanidins, were predicted to inhibit TcAK activity. Here, it was demonstrated that the anthocyanidin delphinidin showed a non-competitive inhibition mechanism of TcAK (Ki arginine = 1.32 µM and Ki ATP = 500 µM). Molecular docking simulations predicted that delphinidin occupies part of the ATP/ADP pocket, more specifically the one that binds the ribose phosphate, and molecular dynamics simulations confirmed the amino acids involved in binding. Delphinidin exerted trypanocidal activity over T. cruzi trypomastigotes with a calculated IC50 of 19.51 µM. Anthocyanidins are low-toxicity natural products which can be exploited for the development of trypanocidal drugs with less secondary effects than those currently used for the treatment of Chagas disease.


Asunto(s)
Antocianinas , Arginina Quinasa , Enfermedad de Chagas , Tripanocidas , Adenosina Difosfato , Adenosina Trifosfato , Antocianinas/farmacología , Arginina/metabolismo , Arginina Quinasa/antagonistas & inhibidores , Enfermedad de Chagas/tratamiento farmacológico , Simulación del Acoplamiento Molecular , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma cruzi
2.
Molecules ; 25(15)2020 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-32731629

RESUMEN

The aim of this study was to explore the inhibitory potential of apoferritin or apoferritin-capped metal nanoparticles (silver, gold and platinum) against Trypanosomabrucei arginine kinase. The arginine kinase activity was determined in the presence and absence of apoferritin or apoferritin-capped metal nanoparticles. In addition, kinetic parameters and relative inhibition of enzyme activity were estimated. Apoferritin or apoferritin-capped metal nanoparticles' interaction with arginine kinase of T. brucei led to a >70% reduction in the enzyme activity. Further analysis to determine kinetic parameters suggests a mixed inhibition by apoferritin or apoferritin-nanoparticles, with a decrease in Vmax. Furthermore, the Km of the enzyme increased for both ATP and L-arginine substrates. Meantime, the inhibition constant (Ki) values for the apoferritin and apoferritin-nanoparticle interaction were in the submicromolar concentration ranging between 0.062 to 0.168 nM and 0.001 to 0.057 nM, respectively, for both substrates (i.e., L-arginine and ATP). Further kinetic analyses are warranted to aid the development of these nanoparticles as selective therapeutics. Also, more studies are required to elucidate the binding properties of these nanoparticles to arginine kinase of T. brucei.


Asunto(s)
Apoferritinas , Arginina Quinasa , Nanopartículas del Metal , Proteínas Protozoarias , Tripanocidas , Trypanosoma brucei brucei/enzimología , Apoferritinas/química , Apoferritinas/farmacología , Arginina Quinasa/antagonistas & inhibidores , Arginina Quinasa/metabolismo , Nanopartículas del Metal/química , Nanopartículas del Metal/uso terapéutico , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Tripanocidas/química , Tripanocidas/farmacología
3.
J Biomol Struct Dyn ; 37(4): 1022-1042, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-29495913

RESUMEN

Cotton is an economically important crop and its production is challenged by the diversity of pests and related insecticide resistance. Identification of the conserved target across the cotton pest will help to design broad spectrum insecticide. In this study, we have identified conserved sequences by Expressed Sequence Tag profiling from three cotton pests namely Aphis gossypii, Helicoverpa armigera, and Spodoptera exigua. One target protein arginine kinase having a key role in insect physiology and energy metabolism was studied further using homology modeling, virtual screening, molecular docking, and molecular dynamics simulation to identify potential biopesticide compounds from the Zinc natural database. We have identified four compounds having excellent inhibitor potential against the identified broad spectrum target which are highly specific to invertebrates.


Asunto(s)
Arginina Quinasa/antagonistas & inhibidores , Gossypium/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Plaguicidas/química , Animales , Arginina Quinasa/genética , Bases de Datos Farmacéuticas , Gossypium/parasitología , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/genética , Plaguicidas/toxicidad , Conformación Proteica , Spodoptera/enzimología , Spodoptera/genética
4.
Int J Biol Macromol ; 101: 653-659, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28359889

RESUMEN

The ciliate Paramecium tetraurelia contains four arginine kinase genes (AK1-4). We detected cDNA for only three of the AKs (AK1-3) via PCR. Recombinant AK1-4 were expressed in Escherichia coli and their kinetics parameters determined. AK3 showed typical substrate inhibition toward arginine, and enzymatic activity markedly decreased when arginine concentration increased. This is the first example of substrate inhibition in wild-type phosphagen kinases. To explore the substrate inhibition mechanism, site-directed mutations were generated, targeting the amino acid sequence D-D-S-Q-V at positions 77-81 in P. tetraurelia AK3. Among the mutants, substrate inhibition was lost remarkably in the S79A mutant. In spite of high amino acid sequence identity (91%) between P. tetraurelia AK3 and AK4, the enzymatic activity of AK4 was less by 3% than that of AK3. We noticed that the conservative G298 was unusually replaced by R in P. tetraurelia AK4, and we constructed two mutants, R298G/AK4 and G298R/AK3. Enzymatic activity of the former mutant was comparable with that of the wild-type AK3, whereas that of the latter mutant was dramatically reduced. Thus, we concluded that the significantly low activity of P. tetraurelia AK4 is due to the residue R298.


Asunto(s)
Arginina Quinasa/antagonistas & inhibidores , Arginina Quinasa/metabolismo , Inhibidores Enzimáticos/metabolismo , Paramecium tetraurelia/enzimología , Secuencia de Aminoácidos , Arginina Quinasa/química , Arginina Quinasa/genética , Inhibidores Enzimáticos/farmacología , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo
5.
J Exp Zool B Mol Dev Evol ; 326(4): 237-49, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27245369

RESUMEN

Energy metabolism is a key process in larval settlement of barnacles, but the underlying molecular mechanisms remain ambiguous. Arginine kinase (AK) mainly participates in energy metabolism in invertebrates. So far, its roles in barnacles have not been studied. In the present study, we raised an antibody against AK from Amphibalanus amphitrite Darwin to characterize the roles of AK in the larval settlement process. Among the developmental stages, AK was highly expressed during the cypris stage. Along with the aging process in cyprids, the level of AK decreased. The immunostaining results showed that AK was localized to muscular tissues in cyprids, including antennules, antennular muscles, and thoracic limbs. The larval settlement rate decreased and larval movement was inhibited in response to treatments with high concentrations of AK inhibitors (rutin and quercetin). These results demonstrated that AK was involved in the larval settlement of A. amphitrite through mediating energy supply in muscle tissues. Moreover, further analysis indicated that both the p38 MAPK and NO/cGMP pathways positively mediated the expression of AK in cyprids.


Asunto(s)
Thoracica/crecimiento & desarrollo , Animales , Arginina Quinasa/antagonistas & inhibidores , Arginina Quinasa/metabolismo , GMP Cíclico/metabolismo , Larva/enzimología , Larva/crecimiento & desarrollo , Músculos/enzimología , Quercetina/farmacología , Rutina/farmacología , Thoracica/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
6.
Int J Biol Macromol ; 91: 926-33, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27318110

RESUMEN

Arginine kinase plays an important role in cellular energy metabolism and is closely related to the environmental stress response in marine invertebrates. We studied the Cu(2+)-mediated inhibition and aggregation of Sepia pharaonis arginine kinase (SPAK) and found that Cu(2+) markedly inhibited the SPAK activity along with mixed-type inhibition against the arginine substrate and noncompetitive inhibition against the ATP cofactor. Spectrofluorimetry results showed that Cu(2+) induced a tertiary structure change in SPAK, resulting in exposure of the hydrophobic surface and increased aggregation. Cu(2+)-mediated SPAK aggregation followed first-order kinetics consistent with monophasic and a biphasic processes. Addition of osmolytes, including glycine and proline, effectively blocked SPAK aggregation and restored SPAK activity. Our results demonstrated the effects of Cu(2+) on SPAK catalytic function, conformation, and aggregation, as well as the protective effects of osmolytes on SPAK folding. This study provided important insights into the role of Cu(2+) as a negative effector of the S. pharaonis metabolic enzyme AK and the possible responses of cephalopods to unfavorable environmental conditions.


Asunto(s)
Arginina Quinasa/química , Arginina Quinasa/metabolismo , Agregado de Proteínas/efectos de los fármacos , Sepia/enzimología , Adenosina Trifosfato/farmacología , Animales , Arginina Quinasa/antagonistas & inhibidores , Dicroismo Circular , Activación Enzimática/efectos de los fármacos , Glicina/farmacología , Cinética , Prolina/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia
7.
Protein Pept Lett ; 23(6): 508-17, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27030550

RESUMEN

Arginine kinase is an essential enzyme which is closely related to energy metabolism in marine invertebrates. Arginine kinase provides a significant role in quick response to environmental change and stress. In this study, we simulated a tertiary structure of Sepia pharaonis arginine kinase (SPAK) based on the gene sequence and conducted the molecular dynamics simulations between SPAK and Zn(2+). Using these results, the Zn(2+) binding sites were predicted and the initial effect of Zn(2+) on the SPAK structure was elucidated. Subsequently, the experimental kinetic results were compared with the simulation results. Zn(2+) markedly inhibited the activity of SPAK in a manner of non-competitive inhibitions for both arginine and ATP. We also found that Zn(2+) binding to SPAK resulted in tertiary conformational change accompanying with the hydrophobic residues exposure. These changes caused SPAK aggregation directly. We screened two protectants, glycine and proline, which effectively prevented SPAK aggregation and recovered the structure and activity. Overall, our study suggested the inhibitory effect of Zn(2+) on SPAK and Zn(2+) can trigger SPAK aggregation after exposing large extent of hydrophobic surface. The protective effects of glycine and proline against Zn(2+) on SPAK folding were also demonstrated.


Asunto(s)
Arginina Quinasa/antagonistas & inhibidores , Sepia/enzimología , Zinc/química , Secuencia de Aminoácidos , Animales , Arginina Quinasa/genética , Arginina Quinasa/metabolismo , Sitios de Unión , Clonación Molecular , Metabolismo Energético , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Simulación de Dinámica Molecular , Sepia/metabolismo
8.
Int J Biol Macromol ; 87: 498-503, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-26976067

RESUMEN

Arginine kinase catalyzes the reversible transphosphorylation between ADP and phosphoarginine which plays a critical role in the maintenance of cellular energy homeostasis. Arginine kinase from the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, meets the requirements to be considered as a potential therapeutic target for rational drug design including being absent in its mammalian hosts. In this study a group of polyphenolic compounds was evaluated as potential inhibitors of arginine kinase using molecular docking techniques. Among the analyzed compounds with the lowest free binding energy to the arginine kinase active site (<-6.96kcal/mol), resveratrol was chosen for subsequent assays. Resveratrol inhibits 50% of recombinant arginine kinase activity at 325µM. The trypanocidal effect of resveratrol was evaluated on the T. cruzi trypomastigotes bursting from infected CHO K1 cells, with IC50=77µM. Additionally epimastigotes overexpressing arginine kinase were 5 times more resistant to resveratrol compared to controls. Taking into account that: (1) resveratrol is considered as completely nontoxic; (2) is easily accessible due to its low market price; and (3) has as a well-defined target enzyme which is absent in the mammalian host, it is a promising compound as a trypanocidal drug for Chagas disease.


Asunto(s)
Arginina Quinasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Estilbenos/farmacología , Tripanocidas/farmacología , Trypanosoma cruzi/enzimología , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Simulación del Acoplamiento Molecular , Polifenoles/farmacología , Conformación Proteica , Resveratrol , Estilbenos/metabolismo , Tripanocidas/metabolismo , Trypanosoma cruzi/efectos de los fármacos
9.
J Biomol Struct Dyn ; 34(8): 1763-77, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26360528

RESUMEN

Arginine kinase is closely associated with adaptation to environmental stresses such as high salinity and heavy metal ion levels in marine invertebrates. In this study, the effects of Cd(2+) on the cuttlefish Sepia pharaonis' arginine kinase (SPAK) were investigated. SPAK was isolated from the muscles of S. pharaonis and upon further purification, showed a single band on SDS-PAGE. Cd(2+) effectively inactivated SPAK, and the double-reciprocal kinetics indicated that Cd(2+) induced non-competitive inhibition of arginine and ATP. Spectrofluorometry results showed that Cd(2+) induced tertiary structure changes in SPAK with the exposure of hydrophobic surfaces that directly induced SPAK aggregation. The addition of osmolytes, glycine, and proline successfully blocked SPAK aggregation and restored the conformation and activity of SPAK. Molecular dynamics simulations involving SPAK and Cd(2+) showed that Cd(2+) partly blocks the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd(2+)-induced inactivation of SPAK. These results demonstrate the effect of Cd(2+) on SPAK enzymatic function and unfolding, including aggregation and the protective effects of osmolytes on SPAK folding. This study provides concrete evidence of the toxicity of Cd(2+) in the context of the metabolic enzyme SPAK, and it illustrates the toxic effects of heavy metals and detoxification mechanisms in cuttlefish.


Asunto(s)
Arginina Quinasa/química , Cadmio/química , Decapodiformes/enzimología , Modelos Moleculares , Conformación Molecular , Pliegue de Proteína , Secuencia de Aminoácidos , Animales , Arginina Quinasa/antagonistas & inhibidores , Arginina Quinasa/aislamiento & purificación , Sitios de Unión , Cadmio/toxicidad , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Concentración de Iones de Hidrógeno , Cinética , Simulación de Dinámica Molecular , Concentración Osmolar , Agregado de Proteínas/efectos de los fármacos , Unión Proteica , Temperatura
10.
Appl Biochem Biotechnol ; 176(4): 1217-36, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25935224

RESUMEN

We studied the Cu(2+)-mediated inhibition and aggregation of Exopalaemon carinicauda arginine kinase (ECAK). We found that Cu(2+) significantly inactivated ECAK activity and double-reciprocal kinetics demonstrated that Cu(2+) induced noncompetitive inhibition of arginine and ATP (IC50 = 2.27 ± 0.16 µM; K i for arginine = 13.53 ± 3.76; K i for ATP = 4.02 ± 0.56). Spectrofluorometry results showed that Cu(2+) induced ECAK tertiary structural changes including the exposure of hydrophobic surfaces that directly induced ECAK aggregation. The addition of osmolytes such as glycine and proline successfully blocked ECAK aggregation induced by Cu(2+) and recovered ECAK activity. We built a 3D structure for ECAK using the ECAK ORF gene sequence. Molecular dynamics (MD) and docking simulations between ECAK and Cu(2+) were conducted to elucidate the binding mechanisms. The results showed that Cu(2+) blocked the entrance to the ATP active site; these results are consistent with the experimental result that Cu(2+) induced ECAK inactivation. Since arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrates, our study can provide new information about the effect of Cu(2+) on ECAK enzymatic function and unfolding, including aggregation, and the protective effects of osmolytes on ECAK folding to better understand the role of the invertebrate ECAK metabolic enzyme in marine environments.


Asunto(s)
Arginina Quinasa/antagonistas & inhibidores , Proteínas de Artrópodos/antagonistas & inhibidores , Cobre/química , Decápodos/química , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Animales , Arginina/química , Arginina Quinasa/química , Arginina Quinasa/aislamiento & purificación , Proteínas de Artrópodos/química , Proteínas de Artrópodos/aislamiento & purificación , Dominio Catalítico , Cationes Bivalentes , Decápodos/enzimología , Glicina/química , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Prolina/química , Agregado de Proteínas , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína
11.
Infect Disord Drug Targets ; 14(1): 30-6, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25019239

RESUMEN

Trypanosomatids parasites have complex life cycles which involve a wide diversity of milieus with very different physicochemical properties. Arginine kinase is one of the key enzymes, responsible for the parasites' metabolic plasticity, which maintains the cell energy homeostasis during environment changes. Arginine kinase catalyzes the reversible phosphorylation between phosphoarginine and ADP. The phosphagen phosphoarginine sustains high levels of cellular activity until metabolic events, such as glycolysis and oxidative phosphorylation, are switched on. In different unicellular and multicellular organisms including trypanosomatids, it was demonstrated that arginine kinase is an important component in resistance mechanisms to different stress factors, such as reactive oxygen species, trypanocidal drugs, pH and starvation. In addition, few arginine kinase inhibitors were identified during the lasts years, some of them with trypanocidal activity, such as polyphenolic compounds. All these unique features, in addition to the fact that arginine kinase is completely absent in mammals, make this pathway a favorable start point for rational drug design for the treatment of human trypanosomamiases.


Asunto(s)
Arginina Quinasa/antagonistas & inhibidores , Diseño de Fármacos , Terapia Molecular Dirigida , Proteínas Protozoarias/antagonistas & inhibidores , Tripanocidas/uso terapéutico , Trypanosoma/efectos de los fármacos , Tripanosomiasis/tratamiento farmacológico , Animales , Arginina/análogos & derivados , Arginina/metabolismo , Arginina Quinasa/metabolismo , Metabolismo Energético/efectos de los fármacos , Humanos , Compuestos Organofosforados/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma/enzimología , Tripanosomiasis/parasitología
12.
Exp Parasitol ; 134(3): 362-7, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23583781

RESUMEN

Full length cDNA encoding arginine kinases (AK) were cloned from Teladorsagia circumcincta (TcAK) and Haemonchus contortus (HcAK). The TcAK and HcAK cDNA (1080 bp) encoded 360 amino acid proteins. The predicted amino acid sequence showed 99% similarity with each other and 94% with a Caenorhabditis elegans AK. Soluble N-terminal His-tagged AK proteins were expressed in Escherichia coli strain BL21, purified and characterised. All binding sites were completely conserved in both proteins. The recombinant TcAK and HcAK had very similar kinetic properties: K(m) arginine was 0.35 mM, K(m) ATP was 0.8-0.9 mM and the pH optima were pH 7.5. Arginine analogues strongly inhibited recombinant enzyme activities (up to 80%), whilst other amino acids decreased activities by a maximum of 20%. TcAK and HcAK are potential vaccine candidates because of the strong antigenicity of invertebrate phosphagens and kinases and presence in metabolically active parts of the worm.


Asunto(s)
Arginina Quinasa/genética , Haemonchus/enzimología , Trichostrongyloidea/enzimología , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Arginina Quinasa/antagonistas & inhibidores , Arginina Quinasa/química , Sitios de Unión , Clonación Molecular , ADN Complementario/genética , ADN de Helmintos/genética , Electroforesis en Gel de Poliacrilamida , Haemonchus/genética , Proteínas del Helminto/química , Proteínas del Helminto/genética , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de Secuencia , Trichostrongyloidea/genética
13.
J Sci Food Agric ; 93(2): 245-53, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22674459

RESUMEN

BACKGROUND: Arginine kinase (AK) is expressed in a wide variety of species, including human food sources (seafood) and pests (cockroaches and moths), and has been reported as a novel allergen. However, there has been little research on the allergenicity of AK in crustaceans. In this study the physicochemical properties of AK from mud crab (Scylla paramamosain) were investigated. RESULTS: Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and inhibition enzyme-linked immunosorbent assay revealed that purified AK was unstable in thermal processing and in acid buffer. Under simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) conditions, purified AK was much more readily degraded by pepsin than by trypsin or chymotrypsin. The unpurified AK in crab myogen degraded more markedly than purified AK. In addition, in two-phase gastrointestinal digestion, AK was rapidly degraded by pepsin but resistant to trypsin and chymotrypsin digestion, while tropomyosin derived from mud crab was resistant to pepsin digestion but digested readily by trypsin or chymotrypsin. Further study of serum samples obtained from crab-allergic human patients indicated that the allergenicity of AK was markedly reduced by digestion with SGF but not SIF. CONCLUSION: AK is an important food allergen despite its unstable physicochemical properties of digestibility.


Asunto(s)
Alérgenos/química , Arginina Quinasa/química , Proteínas de Artrópodos/química , Braquiuros/química , Mariscos/análisis , Alérgenos/efectos adversos , Alérgenos/aislamiento & purificación , Alérgenos/metabolismo , Animales , Arginina Quinasa/antagonistas & inhibidores , Arginina Quinasa/aislamiento & purificación , Arginina Quinasa/metabolismo , Proteínas de Artrópodos/antagonistas & inhibidores , Proteínas de Artrópodos/aislamiento & purificación , Proteínas de Artrópodos/metabolismo , Braquiuros/enzimología , Braquiuros/crecimiento & desarrollo , Fenómenos Químicos , China , Proteínas en la Dieta/análisis , Proteínas en la Dieta/antagonistas & inhibidores , Proteínas en la Dieta/aislamiento & purificación , Proteínas en la Dieta/metabolismo , Digestión , Estabilidad de Enzimas , Hipersensibilidad a los Alimentos/sangre , Hipersensibilidad a los Alimentos/inmunología , Jugo Gástrico/enzimología , Jugo Gástrico/metabolismo , Calor , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina E/metabolismo , Fenómenos Mecánicos , Modelos Moleculares , Pepsina A/metabolismo , Estructura Terciaria de Proteína , Proteolisis , Mariscos/efectos adversos
14.
Int J Biol Macromol ; 49(5): 985-91, 2011 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-21889534

RESUMEN

Arginine kinase plays a vital role in invertebrate homeostasis by buffering ATP concentrations. Arginine kinase might serve as a target for environmentally friendly insect-selective pesticides, because it differs notably from its counterpart in vertebrates, creatine kinase. In this study, two members of the flavonoid family, quercetin (QU) and luteolin (LU), were identified as novel noncompetitive inhibitors of locust arginine kinase. They were found to have inhibition parameters (K(i)) of 11.2 and 23.9 µM, respectively. By comparing changes in the activity and intrinsic fluorescence of AK, the inhibition mechanisms of these flavonoids were found to involve binding to Trp residues in the active site. This was determined by examination of the static quenching parameter K(sv). The main binding forces between flavonoids and AK were found to be hydrophobic based on the thermodynamic parameters of changes in enthalpy (ΔH), entropy (ΔS), and free energy (ΔG) and on docking simulation results. Molecular docking analyses also suggested that flavonoids could bind to the active site of AK and were close to the Trp 221 in active site. Molecular simulation results mimic the experimental results, indicated that QU had a lower binding energy and a stronger inhibitory effect on AK than LU, suggesting that the extra hydroxyl group in QU might increase binding ability.


Asunto(s)
Arginina Quinasa/antagonistas & inhibidores , Flavonoides/farmacología , Saltamontes/efectos de los fármacos , Control de Insectos/métodos , Proteínas de Insectos/antagonistas & inhibidores , Insecticidas , Quercetina/farmacología , Animales , Arginina Quinasa/química , Arginina Quinasa/metabolismo , Dominio Catalítico , Dicroismo Circular , Creatina Quinasa/química , Creatina Quinasa/metabolismo , Saltamontes/enzimología , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Invertebrados , Cinética , Luteolina/farmacología , Simulación de Dinámica Molecular , Unión Proteica/efectos de los fármacos , Soluciones , Espectrometría de Fluorescencia , Termodinámica , Triptófano/metabolismo , Vertebrados
15.
J Biomol Struct Dyn ; 27(1): 59-64, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19492863

RESUMEN

We studied the effects of Ag(+) on arginine kinase (AK) from Fenneropenaeus chinensis. Ag(+) inactivated the activity of AK in a dose dependent manner (IC(50) = 15 microM). Kinetic studies showed that the inactivation of AK by Ag(+) was reversible and occurred in a noncompetitive inhibition manner (K(i) = 2.8 microM). Spectroflurorimetry results showed that Ag(+) did not induce conspicuous tertiary structural changes in AK at the corresponding concentration ranges of inactivation studies. However, the secondary structure measured by circular dichroism was slightly changed by Ag(+). Taken together, these data suggest that the active site of AK is flexible, with the complete loss of activity occurring prior to significant changes in overall structures. Our study provides important insight into the inhibitory mechanism of Ag(+) on AK and increases our understanding of the influence of Ag+ on the mechanism of this metabolic enzyme.


Asunto(s)
Arginina Quinasa/antagonistas & inhibidores , Arginina Quinasa/metabolismo , Decápodos/enzimología , Inhibidores Enzimáticos/metabolismo , Plata/metabolismo , Animales , Arginina Quinasa/química , Estructura Terciaria de Proteína
16.
Int J Biol Macromol ; 44(2): 149-55, 2009 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-19100283

RESUMEN

Arginine kinase (AK; EC 2.7.3.3) is a key enzyme in the cellular energy metabolism of insects. Screening on potential effective inhibitors of AK may provide a pathway for novel, environmentally friendly insecticides. The results in this study indicated that rutin, as a noncompetitive inhibitor, interacts with AK mainly by a hydrophobic force forming an intermolecular complex with AK, which is according to the thermodynamic parameters obtained. Using a flexible docking method (AutoDock) the interaction between rutin and AK were further analyzed, which suggested in order to screen effective inhibitors, flexible active sites of AK (Ser63, Gly64, Val65, Tyr68) should be taken in account.


Asunto(s)
Arginina Quinasa/antagonistas & inhibidores , Simulación por Computador , Modelos Moleculares , Rutina/farmacología , Animales , Sitios de Unión , Saltamontes/enzimología , Concentración de Iones de Hidrógeno/efectos de los fármacos , Cinética , Rutina/química , Espectrometría de Fluorescencia , Temperatura , Termodinámica
17.
Int J Biol Macromol ; 41(5): 564-71, 2007 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-17765964

RESUMEN

Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (DeltaH degrees ) was 12.38kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK.


Asunto(s)
Arginina Quinasa/química , Arginina Quinasa/metabolismo , Arginina , Cisteína , Animales , Arginina Quinasa/antagonistas & inhibidores , Sitios de Unión , Ácido Ditionitrobenzoico/farmacología , Ditiotreitol/farmacología , Inhibidores Enzimáticos/farmacología , Saltamontes/enzimología , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Cinética , Espectrofotometría , Termodinámica
18.
Recent Pat Antiinfect Drug Discov ; 2(1): 19-51, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18221162

RESUMEN

Chagas disease or American Trypanosomiasis, a parasitic infection typically spread by triatomine bugs, affects millions of people throughout Latin America. Current chemotherapy based on the nitroaromatic compounds, benznidazole and nifurtimox provides unsatisfactory results and suffers from considerable side effects and low efficacy. Therefore, there is an urgent need for new drugs to treat this neglected disease. Over the last two decades, new advances and understanding in the biology and the biochemistry of Trypanosoma cruzi has allowed the identification of multiple targets for Chagas disease chemotherapy. This review summarizes antichagasic agents obtained based on i) target metabolic biochemical pathways or parasite specific enzymes, ii) natural products and its derivatives, iii) design and synthesis of lead compounds. Related patents filed and issued from 2000 to early 2006 are also discussed. Most of them claimed inhibitors on specific parasite targets such as cysteine proteinase, sterol biosynthesis, protein farnesyltransferase, etc. Particularly, those related to cysteine proteinase inhibitors were the most represented. Natural products also displayed many anti-T cruzi lead compounds. In addition, a few patents claiming natural or synthetic compounds with antichagasic activity, disclosed no specific target. However, only a small proportion of all these patents displayed specific data of biological trypanocidal activity.


Asunto(s)
Enfermedad de Chagas/tratamiento farmacológico , Tripanocidas/uso terapéutico , Trypanosoma cruzi/efectos de los fármacos , Animales , Arginina Quinasa/antagonistas & inhibidores , Productos Biológicos/química , Productos Biológicos/farmacología , Enfermedad de Chagas/parasitología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Ergosterol/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Lípidos/biosíntesis , Orgánulos/efectos de los fármacos , Patentes como Asunto , Vía de Pentosa Fosfato/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéutico , Purinas/farmacología , Ácidos Siálicos/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Terpenos/síntesis química , Terpenos/farmacología , Tripanocidas/farmacología , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/metabolismo
19.
Int J Biol Macromol ; 40(1): 15-21, 2006 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-16828861

RESUMEN

The aspartic acid (Asp)-induced unfolding and the salt-induced folding of arginine kinase (AK) were studied in terms of enzyme activity, intrinsic fluorescence emission spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra and far-UV circular dichroism (CD) spectra. The results showed that Asp caused inactivation and unfolding of AK with no aggregation during AK denaturation. The unfolding of the whole molecule and the inactivation of AK in different Asp concentrations were compared. Much lower Asp concentration was required to induce inactivation than to produce significant conformational changes of the enzyme molecule. However, with further addition of Asp, the molar ellipticity at 222 and 208 nm, the wavelength shift and the emission intensity of ANS hardly changed. Asp denatured AK was reactivated by dilution. In addition, potassium chloride (KCl) induced the molten globule state with a compact structure after AK was denatured with 7.5 mM Asp. These results collectively elucidate the osmotic effect of Asp anions for the molten globule formed during unfolding process. They also suggest that the effect of Asp differed from that of other denaturants such as guanidine hydrochloride or urea during AK folding. The molten globule state indicates that intermediates exist during AK folding.


Asunto(s)
Arginina Quinasa/química , Penaeidae/enzimología , Animales , Arginina Quinasa/antagonistas & inhibidores , Arginina Quinasa/metabolismo , Ácido Aspártico/farmacología , Dicroismo Circular , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Complejos Multiproteicos , Cloruro de Potasio/farmacología , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína , Espectrometría de Fluorescencia
20.
Arch Insect Biochem Physiol ; 57(4): 166-77, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15540275

RESUMEN

The kinetic mechanism and evaluation of several potential inhibitors of purified arginine kinase from the cockroach (Periplanta americana) were investigated. This monomeric phosphagen kinase is important in maintaining ATP levels during the rapid energy demands of muscle required for contraction and motility. Analysis reveals the following dissociation constants (mM) for the binary complex: E.Arg P-->E+Arg P, K=1.0; E.Arg-->E+Arg, K=0.45; E.MgATP-->E+MgATP, K=0.17; E.MgADP-->E+MgADP, K=0.12; and the ternary complex: Arg P.E.MgADP-->E.MgADP+Arg P, K=0.94; Arg.E.MgATP-->E.MgATP+Arg, K=0.49; MgATP.Enz.Arg-->E.Arg+MgATP, K=0.14; MgADP.E.Arg P-->E.Arg P+MgADP, K=0.09. For a particular substrate, the ratio of the dissociation constants for the binary to ternary complex is close to one, indicating little, if any, cooperativity in substrate binding for the rapid equilibrium, random addition mechanism. The time course of the arginine kinase reaction exhibits a pronounced curvature, which, as described for enzyme from other sources, is attributed to formation of an inhibitory catalytic dead-end complex, MgADP.E.Arg. The curvature is accentuated by the addition of monovalent anions, including borate, thiocyanate, and, most notably, nitrite and nitrate. This effect is attributed to stabilization of the dead-end complex through formation of a transition state analog. However, the substantial decrease in initial velocity (92%) caused by nitrate is due to an additional inhibitory effect, further characterized as non-competitive inhibition (Ki=8.0 mM) with the substrate L-arginine. On the other hand, borate inhibition of the initial velocity is only 30% with significant subsequent curvature, suggesting that this anion functions as an inhibitor mainly by formation of a transition state analog. However, some component of the borate inhibition appears to be mediated by an apparent partial competitive inhibition with L-arginine. D-arginine is not a substrate for arginine kinase from the cockroach, but is an effective competitive inhibitor with a Ki=0.31 mM. L-Canavanine is a weak substrate for arginine kinase (Km=6.7 mM) with a Vmax for the pure enzyme that is approximately one-third that of L-arginine. However, initial velocity experiments of substrate mixtures suggest that competition between L-canavanine and L-arginine may not be a simple summation effect and may involve a structural modification. Sensitivity of arginine kinase activity to D-arginine as well as nitrate and borate anions, coupled with the fact that L-arginine is an essential amino acid for the cockroach, suggest that arginine kinase could be a useful chemotherapeutic target for the control of cockroach proliferation.


Asunto(s)
Arginina Quinasa/antagonistas & inhibidores , Arginina Quinasa/metabolismo , Periplaneta/enzimología , Animales , Arginina/metabolismo , Boratos/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Nitratos/metabolismo , Unión Proteica , Espectrofotometría
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