RESUMEN
Elevation of intracellular Zn2+ following ischemia contributes to cell death by affecting mitochondrial function. Zn2+ is a differential regulator of the mitochondrial enzyme lipoamide dehydrogenase (LADH) at physiological concentrations (Ka = 0.1 µM free zinc), inhibiting lipoamide and accelerating NADH dehydrogenase activities. These differential effects have been attributed to coordination of Zn2+ by LADH active-site cysteines. A detailed kinetic mechanism has now been developed for the diaphorase (NADH-dehydrogenase) reaction catalyzed by pig heart LADH using 2,6-dichlorophenol-indophenol (DCPIP) as a model quinone electron acceptor. Anaerobic stopped-flow experiments show that two-electron reduced LADH is 15-25-fold less active towards DCPIP reduction than four-electron reduced enzyme, or Zn2+-modified reduced LADH (the corresponding values of the rate constants are (6.5 ± 1.5) × 103 M-1·s-1, (9 ± 2) × 104 M-1·s-1, and (1.6 ± 0.5) × 105 M-1·s-1, respectively). Steady-state kinetic studies with different diaphorase substrates show that Zn2+ accelerates reaction rates exclusively for two-electron acceptors (duroquinone, DCPIP), but not for one-electron acceptors (benzoquinone, ubiquinone, ferricyanide). This implies that the two-electron reduced form of LADH, prevalent at low NADH levels, is a poor two-electron donor compared to the four-electron reduced or Zn2+-modified reduced LADH forms. These data suggest that zinc binding to the active-site thiols switches the enzyme from one- to two-electron donor mode. This zinc-activated switch has the potential to alter the ratio of superoxide and H2O2 generated by the LADH oxidase activity.
Asunto(s)
Dihidrolipoamida Deshidrogenasa/metabolismo , Electrones , Miocardio/metabolismo , NADH Deshidrogenasa/metabolismo , Zinc/metabolismo , 2,6-Dicloroindofenol/metabolismo , Animales , Dominio Catalítico , Escherichia coli/enzimología , Peróxido de Hidrógeno/metabolismo , Cinética , Oxidación-Reducción , Superóxidos/metabolismo , Porcinos , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
Bacteriophage enzyme preparations exolysin and endolysin were studied. Exolysin (a phage-associated enzyme) was obtained from tail fraction and endolysin from phage-free cytoplasmic fraction of disintegrated Salmonella enteritidis cells. A new method for purification of these enzymes was developed, and their molecular masses were determined. The main catalytic properties of the studied enzymes (pH optimum and specificity to bacterial substrates) were found to be similar. Both enzymes lyse Escherichia coli cells like chicken egg lysozyme, but more efficiently lyse S. enteritidis cells and cannot lyse Micrococcus luteus, a good substrate for chicken egg lysozyme. Similar properties of exolysin and endolysin suggest that these enzymes are structurally similar or even identical.
Asunto(s)
Endopeptidasas/química , Fagos de Salmonella/enzimología , Proteínas Virales/química , Animales , Biocatálisis , Pollos , Endopeptidasas/aislamiento & purificación , Endopeptidasas/metabolismo , Escherichia coli/metabolismo , Muramidasa/metabolismo , Salmonella enteritidis/efectos de los fármacos , Especificidad por Sustrato , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismoRESUMEN
To simulate in vitro the conditions under which enzymes act in vivo, enzyme molecules have been entrapped in hydrated reverse micelles of a surfactant in organic solvents. In this system the catalytic activity of one of the enzymes studied (peroxidase) became much higher than in water, and the specificity of the other enzyme (alcohol dehydrogenase) was dramatically altered.
Asunto(s)
Catálisis , Enzimas/metabolismo , Agua , Oxidorreductasas de Alcohol/metabolismo , Cinética , Micelas , Peroxidasas/metabolismo , SolventesRESUMEN
As one of the most devastating forms of trauma, spinal cord injury (SCI) remains a challenging clinical problem. The secondary processes associated with the primary injury, such as overproduction of reactive oxygen species (ROS) and inflammation, lead to concomitant compression of the injured spinal cord and neuronal death. Delivery of copper-zinc superoxide dismutase (SOD1), an efficient ROS scavenger, to the site of injury can mitigate SCI-induced oxidative stress and tissue damage. Towards this goal catalytically active nanoformulations of SOD1 ("nanozymes") are developed as a modality for treatment of SCI. Along with the cross-linked polyion complex of SOD1 with polycation poly(ethylene glycol) (PEG)-polylysine (single-coat (SC) nanozyme), we introduce for the first time the chemically cross-linked multilayer polyion complex in which SOD1 is first incorporated into a polyion complex with polycation, then coated by anionic block copolymer, PEG-polyglutamic acid (double-coat (DC) nanozyme). We developed DC nanozymes with high enzymatic activity and ability to retain and protect SOD1 under physiological conditions. Pharmacokinetic study revealed that DC nanozymes significantly prolonged circulation of active SOD1 in the blood stream compared to free SOD1 or SC nanozymes (half-life was 60 vs 6min). Single intravenous injection of DC nanozymes (5kU of SOD1/kg) improved the recovery of locomotor functions in rats with moderate SCI, along with reduction of swelling, concomitant compression of the spinal cord and formation of post-traumatic cysts. Thus, based on the testing in a rodent model the SOD1 DC nanozymes are promising modality for scavenging ROS, decreasing inflammation and edema, and improving recovery after SCI.
Asunto(s)
Nanopartículas/administración & dosificación , Traumatismos de la Médula Espinal/tratamiento farmacológico , Superóxido Dismutasa-1/administración & dosificación , Enfermedad Aguda , Animales , Femenino , Locomoción/efectos de los fármacos , Masculino , Polímeros/administración & dosificación , Polímeros/farmacocinética , Ratas Sprague-Dawley , Ratas Wistar , Traumatismos de la Médula Espinal/fisiopatología , Superóxido Dismutasa-1/farmacocinéticaRESUMEN
Lytic transglycosylases are abundant peptidoglycan lysing enzymes that degrade the heteropolymers of bacterial cell walls in metabolic processes or in the course of a bacteriophage infection. The conventional catalytic mechanism of transglycosylases involves only the Glu or Asp residue. Endolysin gp144 of Pseudomonas aeruginosa bacteriophage phiKZ belongs to the family of Gram-negative transglycosylases with a modular composition and C-terminal location of the catalytic domain. Glu115 of gp144 performs the predicted role of a catalytic residue. However, replacement of this residue does not completely eliminate the activity of the mutant protein. Site-directed mutagenesis has revealed the participation of Tyr197 in the catalytic mechanism, as well as the presence of a second active site involving Glu178 and Tyr147. The existence of the dual active site was supported by computer modeling and monitoring of the molecular dynamics of the changes in the conformation and surface charge distribution as a consequence of point mutations.
RESUMEN
Micellar enzymology, a new trend in molecular biology, studies catalysis by enzymes entrapped in hydrated reversed micelles composed of surfactants (phospholipids, detergents) in organic solvents. The key research problems of micellar enzymology and its relation to enzyme membranology are discussed.
Asunto(s)
Enzimas/fisiología , Técnicas In Vitro , Cinética , Sustancias Macromoleculares , Membranas/enzimología , Micelas , Solubilidad , Especificidad por Sustrato , Termodinámica , AguaRESUMEN
1. The possibility of stabilizing water-soluble enzymes against the inactivation action of organic solvents by means of surfactants has been studied. Several enzymes (alpha-chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), pyrophosphatase (EC 3.6.1.1), peroxidase (EC 1.11.1.7), lactate dehydrogenase (EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.40)) were used to demonstrate that enzymes can be entrapped into reversed micelles formed by surfactants (Aerosol OT, cetyltrimethylammonium bromide, Brij 56) in an organic solvent (benzene, chloroform, octane, cyclohexane). The enzymes solubilized in this way retain their catalytic activity and substrate specificity. 2. A kinetic theory has been put forward that describes enzymatic reactions occurring in a micelle-solvent pseudobiphasic system. In terms of this theory, an explanation is given for the experimental dependence of the Michaelis-Menten equation parameters on the concentrations of the components of a medium (water, organic solvent, surfactant) and also on the combination of the signs of charges in the substrate molecule and on interphase (++, +-, --). 3. The results obtained by us may prove important for applications of enzymes in organic synthesis and for studying the state and role of water in the structure of biomembranes and active centres of enzymes.
Asunto(s)
Coloides , Enzimas/metabolismo , Micelas , Solventes/farmacología , Tensoactivos/farmacología , Benceno/farmacología , Inhibidores Enzimáticos , Cinética , Modelos Químicos , AguaRESUMEN
A heterodimeric enzyme (gamma-glutamyltransferase) was studied in the reversed micellar medium of Aerosol OT (AOT) in octane. As was shown earlier, the size (radius) of inner cavity of the AOT-reversed micelles is determined by their hydration degree, i.e., [H2O]/[AOT] molar ratio, in the system. Owing to this, the dependence of hydrolytic, transpeptidation and autotranspeptidation activities of the enzyme on the hydration degree was investigated using L- and D-isomers of gamma-glutamyl(3-carboxy-4-nitro)anilide and glycylglycine as substrates. For all of the reaction types, the observed dependences are curves with three optima. The optima are found at the hydration degrees, [H2O]/[AOT] = 11, 17 and 26 when the inner cavity radii of reversed micelles are equal to the size of light (Mr 21,000) and heavy (Mr 54,000) subunits of gamma-glutamyltransferase, and to their dimer (Mr 75,000), respectively. Ultracentrifugation experiments showed that a change of the hydration degree resulted in a reversible dissociation of the enzyme to light and heavy subunits. The separation of light and heavy subunits of gamma-glutamyltransferase formed in reversed micelles was carried out and their catalytic properties were studied. The two subunits catalyze hydrolysis and transpeptidation reactions; autotranspeptidation reaction is detected only in the case of the heavy subunit. These findings imply that the reversed micelles of surfactants in organic solvents function as the matrices with adjustable size permitting to regulate the supramolecular structure and the catalytic activity of oligomeric enzymes.
Asunto(s)
Coloides , Micelas , gamma-Glutamiltransferasa/metabolismo , Catálisis , Centrifugación , Ácido Dioctil Sulfosuccínico , Modelos Químicos , Complejos Multienzimáticos , Octanos , Solventes , Tensoactivos , gamma-Glutamiltransferasa/análisisRESUMEN
Using reversed micelles of surfactants solvated by water-organic cosolvent mixtures as a matrix for enzyme entrapping, it is possible to fix the highly reactive alpha-chymotrypsin form. The reactivity of alpha-chymotrypsin towards nonspecific substrates increases to the extent comparable with that observed in reactions involving specific substrates.
Asunto(s)
Quimotripsina , Animales , Bovinos , Quimotripsina/metabolismo , Técnicas In Vitro , Cinética , Micelas , Movimiento (Física) , Páncreas/enzimología , Conformación Proteica , Solventes , Relación Estructura-ActividadRESUMEN
A comparative study of native and artificially glycosylated alpha-chymotrypsin in reversed micelles of Aerosol OT in octane was carried out. D-Glucosamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were used as modifying agents to yield glycosylated enzyme. Unlike the native alpha-chymotrypsin, the modified protein tended to form reversible oligomeric structures, revealed by the appearance of an additional maximum (characteristic of dimeric forms of protein functioning) as a result of the enzyme catalytic activity being dependent on the AOT hydration degree. Dependence of the enzyme catalytic activity on the surfactant concentration in the case of the modified enzyme was similar to that of glycoproteins, and suggests its membrane affinity. The role of carbohydrate moieties in the functioning of glycoproteins is discussed.
Asunto(s)
Quimotripsina/química , Quimotripsina/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Animales , Bovinos , Glucosamina , Glicosilación , Indicadores y Reactivos , Cinética , Sustancias Macromoleculares , Páncreas/enzimologíaRESUMEN
The phenomenon of regulation of the catalytic activity of enzymes via changing their oligomeric composition in the system of reversed micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in octane was studied using alpha-chymotrypsin (CT) from bovine brain and alkaline phosphatase (AP) from calf intestinal mucosa. The dependences of the enzyme catalytic activity on the AOT hydration degree (Wo = [H2O]/[AOT]), the parameter determining the radius (rc) of the inner cavity of micelles, usually represent the bell-shaped curves. The maximal catalytic activity is observed at such Wo when rc is equal to the size of the enzyme molecule. The position of this maximum strictly correlates with the enzyme oligomeric composition. Thus, in the case of CT this is observed at Wo = 12 when rc is equal to the radius (rp) of the CT globule. In the case of artificially produced conjugate containing six cross-linked CT molecules, this is observed at Wo = 43 when rc is equal to the radius of the sphere surrounding the absolute octahedron composed of six CT globules. The dependence of the catalytic activity of AP on Wo represents a curve with two maxima that are observed when rc is equal to rp of either AP monomer (Wo = 17) or AP dimer (Wo = 25). Ultracentrifugation experiments revealed that variation of Wo causes a change in the oligomeric composition of AP - its transition from monomeric (Wo less than 20) to dimeric form (Wo greater than 20). Hence, the observed maxima correspond to functioning of different oligomeric forms of AP.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Quimotripsina/metabolismo , Fosfatasa Alcalina/química , Animales , Catálisis , Bovinos , Quimotripsina/química , Concentración de Iones de Hidrógeno , Mucosa Intestinal/enzimología , Sustancias Macromoleculares , Micelas , Estructura Molecular , Octanos , Páncreas/enzimología , AguaRESUMEN
The properties of penicillin acylase from E. coli solubilized by hydrated reversed micelles (RM) of Aerosol OT in octane were studied. The dependence of catalytic activity on the hydration degree, a parameter which determines the size of the micelle inner cavity, has a curve with three optima, each one corresponding to the enzyme functioning either in a dimer form (wo = 23) or in a form of separate subunits, a heavy one, beta, and a light one, alpha (wo = 20 and 14, respectively). The reversible dissociation of the enzyme was confirmed by ultracentrifugation followed by electrophoresis.
Asunto(s)
Escherichia coli/enzimología , Penicilina Amidasa/química , Penicilina Amidasa/metabolismo , Ácido Dioctil Sulfosuccínico , Electroforesis en Gel de Poliacrilamida , Cinética , Sustancias Macromoleculares , Micelas , Octanos/farmacología , Penicilina Amidasa/aislamiento & purificación , Fluoruro de Fenilmetilsulfonilo/farmacología , TensoactivosRESUMEN
Regulation of supra-macromolecular composition and catalytic activity of a heterodimeric enzyme, gamma-glutamyltransferase, in the system of Aerosol OT (sodium bis(2-ethylhexyl) sulfosuccinate) reversed micelles in octane were studied. Variation of the surfactant hydration degree (parameter, determining dimensions of the polar inner cavity of the micelle) causes a reversible dissociation of the enzyme to light and heavy subunits. Both enzyme subunits possess catalytic activity. The light and heavy subunits of the enzyme were separated on a preparative scale in a reversed micelle system using ultracentrifugation. The active centers of gamma-glutamyltransferase were studied using its irreversible inhibitor--AT-125 (L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). Separation of the gamma-glutamyltransferase subunits results in the 'opening' of a new active center located at the heavy subunit. In the dimer form of the enzyme this center is masked and it is not accessible to both substrate and inhibitor molecules.
Asunto(s)
gamma-Glutamiltransferasa/metabolismo , Sitios de Unión , Isoxazoles/farmacología , Cinética , Sustancias Macromoleculares , Micelas , Células Tumorales Cultivadas , gamma-Glutamiltransferasa/antagonistas & inhibidores , gamma-Glutamiltransferasa/aislamiento & purificaciónRESUMEN
alpha-Chymotrypsin (CT) solubilized in reversed micelles of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT) undergoes thermal inactivation and the enzyme stability decreases significantly when temperature increases (25-40 degrees C). The half-life of CT in micelles shows a bell-shaped dependence on the degree of hydration of AOT (wo) analogous to the previously obtained dependence on wo for the enzyme activity. The optima of catalytic activity and thermal stability have been observed under conditions where the diameter of the inner aqueous cavity of the micelle is close to the size of the enzyme molecule (wo = 10). Application of high hydrostatic pressure in the range of 1-1500 atm (bar) stabilizes CT against thermal inactivation at all hydration degrees (wo) from 7 to 20; the stabilization effect is most pronounced under the experimental conditions being far from the optimum for catalytic activity.
Asunto(s)
Quimotripsina/química , Animales , Bovinos , Ácido Dioctil Sulfosuccínico/química , Estabilidad de Enzimas , Calor , Cinética , Micelas , Octanos/química , Tensoactivos/químicaRESUMEN
We have used the enhanced green fluorescent protein (EGFP) to investigate the properties of surfactant-entrapped water pools in organic solvents (reversed micelles) with steady-state and time-resolved fluorescence methods. The surfactant used was sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and the organic solvents were isooctane and (the more viscous) dodecane, respectively. The water content of the water pools could be controlled through the parameter w0, which is the water-to-surfactant molar ratio. With steady-state fluorescence, it was observed that subtle fluorescence changes could be noted in reversed micelles of different water contents. EGFP can be used as a pH-indicator of the water droplets in reversed micelles. Time-resolved fluorescence methods also revealed subtle changes in fluorescence decay times when the results in bulk water were compared with those in reversed micelles. The average fluorescence lifetimes of EGFP scaled with the relative fluorescence intensities. Time-resolved fluorescence anisotropy of EGFP in aqueous solution and reversed micelles yielded single rotational correlation times. Geometrical considerations could assign the observed correlation times to dehydrated protein at low w0 and internal EGFP rotation within the droplet at the highest w0.
Asunto(s)
Ácido Dioctil Sulfosuccínico/metabolismo , Fluorescencia , Proteínas Luminiscentes/química , Micelas , Tensoactivos/metabolismo , Alcanos/metabolismo , Alcanos/farmacología , Animales , Ácido Dioctil Sulfosuccínico/farmacología , Polarización de Fluorescencia , Proteínas Fluorescentes Verdes , Concentración de Iones de Hidrógeno , Cinética , Proteínas Luminiscentes/metabolismo , Octanos/metabolismo , Octanos/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rotación , Solventes , Espectrometría de Fluorescencia , Tensoactivos/farmacología , Agua/metabolismo , Agua/farmacologíaRESUMEN
Inclusion of an oligomeric enzyme, NAD+-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha, into a system of reverse micelles of different sizes resulted in its dissociation into catalytically active heterodimers and subunits, which were characterized in reactions with various substrates. It was found that: 1) the native tetrameric form of this enzyme catalyzes all types of studied reactions; 2) hydrogenase dimer, HoxHY, is a minimal structural unit catalyzing hydrogenase reaction with an artificial electron donor, reduced methyl viologen; 3) all structural fragments containing FMN and NAD+/NADH-binding sites exhibit catalytic activity in diaphorase reactions with one- and two-electron acceptors; 4) small subunits, HoxY and HoxU also exhibit activity in diaphorase reactions with artificial acceptors. These results can be considered as indirect evidence that the second FMN molecule may be associated with one of the small subunits (HoxY or HoxU) of the hydrogenase from R. eutropha.
Asunto(s)
Cupriavidus necator/enzimología , Mononucleótido de Flavina/genética , Micelas , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Catálisis , Inmunoelectroforesis Bidimensional , Peso Molecular , NADH Deshidrogenasa/metabolismo , Relación Estructura-ActividadRESUMEN
Refolded alpha and beta subunits of penicillin acylase were produced by gel-filtration under denaturing conditions followed by removal of urea through dialysis. Preparations of both renatured subunits hidrolysed specific substrate--phenylacetic acid p-nitroanilide in buffer and in the system of Aerosol OT reversed micelles, the alpha subunit being most catalytically active at Wo = 11.9, while the beta subunit--at Wo = 17.5. There was a good correlation between the position of the found maxima, the theoretically calculated optimal hydration degrees as well as the earlier reported profile of enzymatic activity for native enzyme in reversed micelles.
Asunto(s)
Bencenoacetamidas , Ácido Dioctil Sulfosuccínico/química , Octanos/química , Penicilina Amidasa/metabolismo , Tensoactivos/química , Compuestos de Anilina/metabolismo , Tampones (Química) , Catálisis , Cromatografía en Gel , Compuestos Cromogénicos/metabolismo , Ácido Dioctil Sulfosuccínico/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Hidrólisis , Micelas , Penicilina Amidasa/química , Penicilina Amidasa/aislamiento & purificación , Pliegue de Proteína , Especificidad por Sustrato , Tensoactivos/metabolismo , Urea/químicaRESUMEN
Stability of recombinant peroxidase lacking carbohydrate residues on the surface of the protein molecule has been characterized in reversed micelles of Aerosol OT in octane. The enzyme stability was found to depend on the surfactant hydration degree (w0 = [H2O]/[AOT]). Residual activity after 1 h incubation dropped to zero at w0 = 7 but was 54% at w0 = 25. However, the residual activity levels at all values of hydration degree were definitely low compared to that of glycosylated wild-type horseradish peroxidase. The stability of the enzyme apparently depends on the presence of carbohydrate residues. Stabilization of recombinant peroxidase in reversed micellar system involved sugar-containing co-surfactants such as Tweens and Spans is proposed. As an example, addition of 1 mM Span 80 (1% relative to AOT concentration) increased the recombinant peroxidase stability up to that of wild-type peroxidase.
Asunto(s)
Escherichia coli/enzimología , Micelas , Peroxidasa/química , Detergentes , Ácido Dioctil Sulfosuccínico/metabolismo , Estabilidad de Enzimas , Glicosilación , Hexosas , Cinética , Oligosacáridos/farmacología , Peroxidasa/genética , Peroxidasa/metabolismo , Polisorbatos , Proteínas Recombinantes/químicaRESUMEN
Serine-specific irreversible inhibitor phenylmethanesulfonyl fluoride (PMSF) inactivates penicillin acylase subunits, which were chromatographically separated under denaturing conditions and refolded by dialysis, in aqueous solution and Aerosol OT reversed micelles. The activities of both alpha and beta subunits decrease with increasing PMSF concentration but the dependence is no longer linear, in contrast with the native enzyme. The enzyme inactivated in aqueous solution, when solubilized in the micellar system at Wo = 12, exhibits an additional activity, which can be further inhibited by PMSF.