RESUMO
Chemiluminescence (CL) reactions are widely used for the detection and quantification of many types of analytes. Laccase has previously been proposed in CL reactions; however, its light emission behaviour has not been characterized. This study was conducted to characterize the laccase-luminol system, determine its kinetic parameters, and analyze the effects of protein and OH- concentration on the CL signal. Laccase from Coriolopsis gallica was combined with different concentrations of luminol (125 nM to 4 mM), and the enzyme kinetics were evaluated using diverse kinetic models. The laccase-luminol system was able to produce CL without an intermediate molecule, but it exhibited substrate-inhibition behaviour. A two-site random model was used and suggested that when the first luminol molecule was bound to the active site, laccase affinity for the second luminol molecule was increased. This inhibition effect could be avoided using a low luminol concentration. At 5 µM luminol concentration, 1 mg/ml (0.13 U) laccase is needed to achieve nearly 90% of the maximum CL signal, suggesting that the available luminol could not bind to all active sites. Furthermore, the concentration of NaOH negatively affected the CL signal. The laccase-luminol system represents an alternative to existing CL systems, with potential uses in molecular detection and quantification.
Assuntos
Lacase , Luminol , Luminol/química , Lacase/química , Luminescência , Medições LuminescentesRESUMO
Methanogenic archaea have received attention due to their potential use in biotechnological applications such as methane production, so their metabolism and regulation are topics of special interest. When growing in a nutrient-rich medium, these organisms exhibit gluconeogenic metabolism; however, under starvation conditions, they turn to glycolytic metabolism. To date, no regulatory mechanism has been described for this gluconeogenic/glycolytic metabolic switch. Here, we report that adenosine monophosphate (AMP) activates both enzymatic activities of the bifunctional adenosine diphosphate (ADP)-dependent phosphofructokinase/glucokinase from Methanococcus maripaludis (MmPFK/GK). To understand this phenomenon, we performed a comprehensive kinetic characterisation, including determination of the kinetics, substrate inhibition and AMP activation mechanism of this enzyme. We determined that MmPFK/GK has an ordered-sequential mechanism, in which MgADP is the first substrate to bind and AMP is the last product released. The enzyme also displays substrate inhibition by both sugar substrates; we determined that this inhibition occurs through the formation of catalytically nonproductive enzyme complexes caused by sugar binding. For both activities, the AMP activation mechanism occurs primarily through incremental changes in the affinity for the sugar substrate, with this effect being higher in the GK than in the PFK activity. Interestingly, due to the increase in the sugar substrate affinity caused by AMP, an enhancement in the sugar substrate inhibition effect was also observed for both activities, which can be explained by an increase in sugar binding leading to the formation of dead-end complexes. These results shed light on the regulatory mechanisms of methanogenic archaeal sugar metabolism, a phenomenon that has been largely unexplored.
Assuntos
Mathanococcus , Fosfofrutoquinases , Difosfato de Adenosina , Monofosfato de Adenosina , Mathanococcus/genética , AçúcaresRESUMO
Serpins are a superfamily of proteins characterized by their common function as serine protease inhibitors. So far, 36 serpins from nine clades have been identified. These proteins are expressed in all the organs and are involved in multiple important functions such as the regulation of blood pressure, hormone transport, insulin sensitivity, and the inflammatory response. Diseases such as obesity, diabetes, cardiovascular diseases, and kidney disorders are intensively studied to find effective therapeutic targets. Given the serpins' outstanding functionality, the deficiency or overexpression of certain types of serpin has been associated with diverse pathophysiological events. In particular, we focus here on reviewing the studies evaluating the participation of serpins, and particularly SerpinA3, in diverse diseases that occur in relevant organs such as the brain, retinas, corneas, lungs, cardiac vasculature, and kidneys. In this review, we summarize the role of serpins in physiological and pathophysiological processes as well as recent evidence on the crucial role of SerpinA3 in several pathologies. Finally, we emphasize the importance of SerpinA3 in regulating cellular processes such as angiogenesis, apoptosis, fibrosis, oxidative stress, and the inflammatory response.
Assuntos
Doença Crônica , Saúde , Serpinas/metabolismo , Animais , Apoptose , Fibrose , Humanos , Inflamação/metabolismo , Neovascularização Fisiológica , Estresse Oxidativo , Transdução de SinaisRESUMO
In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. Δ1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from Δ1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is an NADPH-dependent cytosolic enzyme with a Kmapp for P5C of 27.7â µM and with a higher expression in the insect-resident form of the parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Kiapp=45±0.7µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model, cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite, and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase.
Assuntos
NADP/metabolismo , Prolina/metabolismo , Pirróis/metabolismo , Trypanosoma cruzi/metabolismo , Citosol/metabolismo , Transporte de Elétrons , Ácido Glutâmico/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Pirrolina Carboxilato Redutases/metabolismoRESUMO
In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. delta1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from delta1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is a NADPH-dependent cytosolic enzyme with a Km app for P5C of 23.9 mM and with a higher expression in the insect-resident form of parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Ki app = 45 ± 0.7 µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase
RESUMO
The influence of ethanol on the degradation kinetics of linear alkyl benzene sulfonate (LAS) and organic matter was investigated using batch experiments with different initial LAS concentrations (8.3 mg L-1 to 66.9 mg L-1) and biomass immobilized on sand. Data were fitted with a substrate inhibition model. Concentrations of 2.4 mg LAS L-1 and 18.9 mg LAS L-1 (without and with ethanol) provided the maximum LAS utilization rate by the biomass (Sbm). For LAS degradation, ethanol addition favored a lower decrease in the specific substrate utilization rate (robs), even at the LAS concentration usually reported as inhibitory (> 14.4 mg L-1). For organic matter degradation, robs was higher with ethanol. Higher biomass differentiation was observed at higher LAS concentrations. With ethanol, microbial selection occurred at LAS concentrations near Sbm. At higher LAS concentrations, the dominance and diversity values did not change significantly with ethanol, whereas without ethanol, their behaviors were irregular.
Assuntos
Ácidos Alcanossulfônicos/metabolismo , Biomassa , Etanol/metabolismo , Esgotos , Eliminação de Resíduos Líquidos , Purificação da Água , Biodegradação AmbientalRESUMO
Background: the growth of world aquaculture has generated important environmental impacts as discard residues that are important sources of protein, which has been used to manufacture low-value products, such as animal food, fish flour and fertilizers. Objectives: to evaluate the effect of enzyme and substrate concentration on the degree of hydrolysis (DH) of proteins in the red tilapia (Oreochromis sp.) viscera (RTV). Methods: the commercial alcalase 2.4 L enzyme was used at different concentrations to hydrolyse the proteins in RTV at 53.5°C and a pH of 9.5 in a 1 L magnetically stirred, jacketed, glass batch reactor connected to an automatic titrator. Each experiment was conducted over 6 h in which every consumed volume of base was recorded every 5 min to determine the corresponding DH at each point. Results: the results indicated that increasing the enzyme concentration produced an increase in the DH and in the reaction rate, while increasing the substrate concentration produced a decrease in both parameters. For this reason, a mathematical model was adjusted for the inhibition of substrate from the exponential kinetic equation d(DH)/dt = a* EXP[-b* (DH)] to explain the behavior of the DH as a function of substrate concentration in this hydrolytic process. The parameters a and b were estimated from a nonlinear regression. Based on these results, the reaction constants were determined as Ks = 456.75 g L-1, K2 = 1.2191 min-1, Kd = 0.2224 min-1, KM = 1.8963 and K3 = 0.1173 L g-1 min-1, which allowed the generation of a good correlation between the predicted and experimental values at the different evaluated operating conditions. This correlation was supported by a low average relative error (ARE) of 3.26%. Conclusion: under evaluated experimental conditions, the kinetics of the hydrolysis reaction followed a substrate inhibition mechanism without product inhibition, which was adjusted through a typical exponential Equation that involves two parameters (a and b) associated with the kinetic constants (Ks, K2, and Kd).
Antecedentes: el crecimiento de la acuicultura en el mundo ha provocado importantes impactos ambientales como el descarte de residuos que son importantes fuentes de proteína, los cuales han sido usados para manufacturar productos de bajo valor tales como: alimento para animales, harina de pescado y fertilizantes. Objetivo: evaluar el efecto de la concentración de enzima y de sustrato sobre el grado de hidrolisis (GH) de las proteínas presentes en las vísceras de tilapia roja (Oreochromis sp.) (VTR). Métodos: se empleó la enzima commercial alcalasa 2.4 L a diferentes concentraciones para hidrolizar las proteínas presentes en la VTR a 53°C y a un pH de 9.5 en un reactor de vidrio de 1 L con chaqueta, magnéticamente agitado y conectado a un titulador automático. Cada experimento se llevó cabo por 6 h registrando cada 5 min el volumen de base cosumido para determinar el grado de hidrolisis correspondiente a cada punto. Resultados: los resultados indicaron que un incremento en la concentración de enzima producía un incremento en el GH y en la velocidad de reacción, mientras que un aumento en la concentración de sustrato provocaba una disminución en ambos parámetros. Por esta razón, se ajustó un modelo matemático para la inhibición de sustrato a partir de la ecuación de cinética exponencial d(GH)/dt = a*EXP[-b*(GH)] para explicar el comportamiento del GH como una función de la concentración de sustrato en este proceso hidrolítico. Los parámetros a y b fueron evaluados mediante una regresión no lineal. Con base en estos resultados, las constantes de reacción fueron determinadas como Ks = 456.75 g L-1, K2 = 1.2191 min-1, Kd = 0.2224 min-1, KM = 1.8963 and K3 = 0.1173 L g-1 min-1, los cuales permitieron obtener una buena correlación entre los valores experimentales y los predichos a las diferentes condiciones de operación. Esta correlación fue soportada por un bajo error medio relativo del 3.26%. Conclusión: bajo las condiciones experimentales evaluadas, la cinética de la reacción de hidrólisis siguió un mecanismo de inhibición por sustrato sin inhibición por producto, el cual fue ajustado mediante una ecuación típica exponencial que involucra dos parámetros (a and b) asociados a las constantes cinéticas Ks, K2, and Kd.
Assuntos
Humanos , Hidrólise , Cinética , Tilápia , Modelos TeóricosRESUMO
We provide initial rate data from enzymatic reaction experiments and tis processing to estimate the kinetic parameters from the substrate uncompetitive inhibition equation using the median method published by Eisenthal and Cornish-Bowden (Cornish-Bowden and Eisenthal, 1974; Eisenthal and Cornish-Bowden, 1974). The method was denominated the direct linear plot and consists in the calculation of the median from a dataset of kinetic parameters Vmax and Km from the Michaelis-Menten equation. In this opportunity we present the procedure to applicate the direct linear plot to the substrate uncompetitive inhibition equation; a three-parameter equation. The median method is characterized for its robustness and its insensibility to outlier. The calculations are presented in an Excel datasheet and a computational algorithm was developed in the free software Python. The kinetic parameters of the substrate uncompetitive inhibition equation Vmax , Km and Ks were calculated using three experimental points from the dataset formed by 13 experimental points. All the 286 combinations were calculated. The dataset of kinetic parameters resulting from this combinatorial was used to calculate the median which corresponds to the statistic estimator of the real kinetic parameters. A comparative statistical analyses between the median method and the least squares was published in Valencia et al. [3].
RESUMO
In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD(+)-dependent oxidation of betaine aldehyde (BAL) catalysed by some aldehyde dehydrogenase (ALDH) 10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse decreases in the NAD(+):NADH ratio. In the present study, we report that the Spinacia oleracea BADH (SoBADH) is reversibly and partially inactivated by BAL in the absence of NAD(+)in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys(450)(SoBADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. It is of interest that, in contrast to Cys(450), the catalytic cysteine (Cys(291)) did not react with BAL in the absence of NAD(+) The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C(450)SSoBADH mutant and the degree of inactivation of the A(441)I and A(441)C mutants corresponds to their very different abilities to bind the trimethylammonium group. Cys(450)and the neighbouring residues that participate in stabilizing the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is their common feature. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to preventing NAD(+)exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL.
Assuntos
Betaína-Aldeído Desidrogenase/química , Betaína/análogos & derivados , Mutação de Sentido Incorreto , Proteínas de Plantas/química , Spinacia oleracea/enzimologia , Substituição de Aminoácidos , Betaína/química , Betaína-Aldeído Desidrogenase/genética , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Proteínas de Plantas/genética , Spinacia oleracea/genéticaRESUMO
The glycolytic enzyme phosphoglycerate kinase (PGK) is present in Trypanosoma cruzi as three isoenzymes, two of them located inside glycosomes (PGKA and PGKC) and another one in the cytosol (PGKB). The three isoenzymes are expressed at all stages of the life cycle of the parasite. A heterologous expression system for PGKA (rPGKA) was developed and the substrate affinities of the natural and recombinant PGKA isoenzyme were determined. Km values measured for 3-phosphoglycerate (3PGA) were 174 and 850 µM, and for ATP 217 and 236 µM, for the natural and recombinant enzyme, respectively. No significant differences were found between the two forms of the enzyme. The rPGKA was inhibited by Suramin with Ki values of 10.08 µM and 12.11 µM for ATP and 3PGA, respectively, and the natural enzyme was inhibited at similar values. A site-directed mutant was created in which the 80 amino acids PGKA sequence, present as a distinctive insertion in the N-terminal domain, was deleted. This internally truncated PGKA showed the same Km values and specific activity as the full-length rPGKA. The natural PGKC isoenzyme was purified from epimastigotes and separated from PGKA through molecular exclusion chromatography and its kinetic characteristics were determined. The Km value obtained for 3PGA was 192 µM, and 10 µM for ATP. Contrary to PGKA, the activity of PGKC is tightly regulated by ATP (substrate inhibition) with a Ki of 270 µM, suggesting a role for this isoenzyme in regulating metabolic fluxes inside the glycosomes.
Assuntos
Metabolismo dos Carboidratos/fisiologia , Fosfoglicerato Quinase/fisiologia , Trypanosoma cruzi/metabolismo , Animais , Western Blotting , Clonagem Molecular , Citosol/enzimologia , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/fisiologia , Cinética , Estágios do Ciclo de Vida , Microcorpos/enzimologia , Fosfoglicerato Quinase/antagonistas & inibidores , Fosfoglicerato Quinase/genética , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Suramina/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
BACKGROUND: Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P2 and by high concentrations of its substrate Fru-1,6-P2. The mechanism that produces substrate inhibition continues to be obscure. METHODS: Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P2 and Fru-1,6-P2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244. RESULTS: The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P2, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a "stapler" that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition. CONCLUSIONS: Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits. GENERAL SIGNIFICANCE: Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.
Assuntos
Biocatálise , Frutose-Bifosfatase/química , Rim/enzimologia , Animais , Sequência de Bases , Sítios de Ligação , Frutose-Bifosfatase/antagonistas & inibidores , Frutose-Bifosfatase/metabolismo , Frutosedifosfatos/química , Dados de Sequência Molecular , Subunidades Proteicas , Especificidade por Substrato , SuínosRESUMO
Autotrophic denitrification is a suitable technology to simultaneously remove oxidised nitrogen compounds and reduced sulphur compounds yielding nitrogen gas, sulphur and sulphate as the main products. In this work, several batch tests were conducted to investigate the cross effect of temperature, pH and free ammonia on the autotrophic denitrification. Denitrification efficiencies above 95% were achieved at 35°C and pH 7.5-8.0 with maximum specific autotrophic denitrifying activities up to 188mgN2g(-1)VSSd(-1). Free ammonia did not show any effect on denitrification at concentrations up to 53mg NH3-NL(-1). Different sulphide concentrations were also tested with stoichiometric nitrite and nitrate concentrations. Sulphide inhibited denitrification at concentrations higher than 200mgS(2-)L(-1). A 50% inhibition was also found at nitrite concentrations above 48mg NO2(-)-NL(-1). The maximum specific activity decreased until a value of 25mgN2g(-1) VSSd(-1) at 232mg NO2(-)-NL(-1). The Haldane model was used to describe denitrification inhibition caused by nitrite. Kinetic parameters determined from the fitting of experimental data were rmax=176mgN2g(-1)VSSd(-1), Ks=10.7mg NO2(-)-NL(-1) and Ki=34.7mg NO2(-)-NL(-1). The obtained model allowed optimising an autotrophic denitrification process by avoiding situations of inhibition and thus obtaining higher denitrification efficiencies.
Assuntos
Amônia/química , Desnitrificação , Modelos Químicos , Sulfetos/química , Processos Autotróficos , Reatores Biológicos , Elétrons , Concentração de Íons de Hidrogênio , Cinética , Nitratos/química , Nitritos/química , Nitrogênio/química , Compostos de Nitrogênio/química , Sulfatos/química , Enxofre/química , TemperaturaRESUMO
The biodegradation kinetics of o-cresol was examined by acclimatized P. putida DSM 548 (pJP4) in batch experiments at varying initial o-cresol concentrations (from 50 to 500 mg/L). The kinetic parameters of o-cresol aerobic biodegradation were estimated by using the Haldane substrate inhibition equation. The biodegradation kinetics of o-cresol was investigated. In batch culture reactors, the Maximum specific growth rate (μmax), Monod constant (Ks) and the inhibition constant (Ki) were established as 0.519 h-1, 223.84 mg/L and 130.883 mg/L, respectively. o-cresol biodegradation in a batch-recirculation bioreactor system by immobilized P. putida was also studied. The recycled packed bed reactor system, which was composed of Ca-alginate beads and pumice on which cells immobilized, has been performed to determine possible stability for further developments.
Assuntos
Biodegradação Ambiental , Cresóis/metabolismo , Pseudomonas putida/química , Reatores Biológicos , Células Imobilizadas , Fenóis/metabolismo , CinéticaRESUMO
This study evaluated the biodegradation of catechol by a yeast strain of Candida parapsilopsis in standard medium in Erlenmeyer flasks. Results shown that the highest concentration of catechol caused the longer lag period, demonstrating that acclimatized cultures could completely degrade an initial catechol concentration of 910 mg/L within 48 h. Haldane's model validated the experimental data adequately for growth kinetics over the studied catechol concentration ranges of 36 to 910 mg/L. The constants obtained for this model were µmax = 0.246 h-1, Ks = 16.95 mg/L and Ki = 604.85 mg/L.
Neste trabalho foi estudada a biodegradação de catecol em frascos de Erlenmeyers em água residuária sintética pela levedura Candida parapsilopsis. As respostas dos ensaios cinéticos mostraram que altas concentrações de catecol ocasionaram uma fase lag longa para a levedura. Portanto, a aclimatização da cultura de levedura empregada para biodegradação de catecol é de fundamental importância, sendo possível reduzir toda a concentração inicial de catecol da água residuária sintética de 910 mg/L em 48 horas. Os dados experimentais da cinética de biodegradação do catecol foram ajustados pelo modelo de Haldane adequadamente, sobre a faixa de concentração de catecol investigada de 36 a 910 mg/L. Os parâmetros cinéticos obtidos do modelo de Haldane foram: µmax = 0,246 h-1, Ks = 16,95 mg/L e Ki = 604,85 mg/L.