RESUMEN
Archaea are uniquely adapted to thrive in harsh environments, and one of these adaptations involves the archaeal membrane lipids, which are characterized by their isoprenoid alkyl chains connected via ether linkages to glycerol 1-phosphate. The membrane lipids of the thermophilic and acidophilic euryarchaeota Thermoplasma volcanium are exclusively glycerol dibiphytanyl glycerol tetraethers. The first committed step in the biosynthetic pathway of these archaeal lipids is the formation of the ether linkage between glycerol 1-phosphate and geranylgeranyl diphosphate, and is catalyzed by the enzyme geranylgeranylglyceryl phosphate synthase (GGGPS). The 1.72â Å resolution crystal structure of GGGPS from T. volcanium (TvGGGPS) in complex with glycerol and sulfate is reported here. The crystal structure reveals TvGGGPS to be a dimer, which is consistent with the absence of the aromatic anchor residue in helix α5a that is required for hexamerization in other GGGPS homologs; the hexameric quaternary structure in GGGPS is thought to provide thermostability. A phylogenetic analysis of the Euryarchaeota and a parallel ancestral state reconstruction investigated the relationship between optimal growth temperature and the ancestral sequences. The presence of an aromatic anchor residue is not explained by temperature as an ecological parameter. An examination of the active site of the TvGGGPS dimer revealed that it may be able to accommodate longer isoprenoid substrates, supporting an alternative pathway of isoprenoid membrane-lipid synthesis.
Asunto(s)
Transferasas Alquil y Aril/química , Proteínas Arqueales/química , Dimetilaliltranstransferasa/química , Éteres Fosfolípidos/metabolismo , Thermoplasma/enzimología , Dominio Catalítico , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
Novel structural superfamilies can be identified among the large number of protein structures deposited in the Protein Data Bank based on conservation of fold in addition to conservation of amino acid sequence. Since sequence diverges more rapidly than fold in protein Evolution, proteins with little or no significant sequence identity are occasionally observed to adopt similar folds, thereby reflecting unanticipated evolutionary relationships. Here, we review the unique alpha/beta fold first observed in the manganese metalloenzyme rat liver arginase, consisting of a parallel eight-stranded beta-sheet surrounded by several helices, and its evolutionary relationship with the zinc-requiring and/or iron-requiring histone deacetylases and acetylpolyamine amidohydrolases. Structural comparisons reveal key features of the core alpha/beta fold that contribute to the divergent metal ion specificity and stoichiometry required for the chemical and biological functions of these enzymes.
Asunto(s)
Arginasa/química , Arginasa/genética , Secuencia de Aminoácidos , Aminohidrolasas/química , Aminohidrolasas/genética , Aminohidrolasas/fisiología , Animales , Arginasa/fisiología , Sitios de Unión , Evolución Molecular , Histona Desacetilasas/química , Histona Desacetilasas/genética , Histona Desacetilasas/fisiología , Humanos , Hígado/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Ratas , Homología de Secuencia de AminoácidoRESUMEN
The x-ray crystal structure of recombinant trichodiene synthase from Fusarium sporotrichioides has been determined to 2.5-A resolution, both unliganded and complexed with inorganic pyrophosphate. This reaction product coordinates to three Mg(2+) ions near the mouth of the active site cleft. A comparison of the liganded and unliganded structures reveals a ligand-induced conformational change that closes the mouth of the active site cleft. Binding of the substrate farnesyl diphosphate similarly may trigger this conformational change, which would facilitate catalysis by protecting reactive carbocationic intermediates in the cyclization cascade. Trichodiene synthase also shares significant structural similarity with other sesquiterpene synthases despite a lack of significant sequence identity. This similarity indicates divergence from a common ancestor early in the evolution of terpene biosynthesis.
Asunto(s)
Liasas de Carbono-Carbono/química , Secuencia de Aminoácidos , Liasas de Carbono-Carbono/metabolismo , Cristalografía por Rayos X , Difosfatos/química , Difosfatos/metabolismo , Fusarium/enzimología , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Terpenos/metabolismoRESUMEN
Intermolecular interactions of eleven different fluoroaromatic inhibitors are probed within the scaffolding of the crystal lattice of Phe-131-->Val carbonic anhydrase II. The degree and pattern of fluorine substitution on the inhibitor benzyl ring modulate its size, shape, and electronic character. In turn, these properties affect the geometry of intermolecular interactions between the fluoroaromatic rings of two different inhibitor molecules bound in the crystal lattice, as determined by X-ray crystallography. Depending on the degree and pattern of fluorine substitution, we observe a face-to-face (aromatic-aromatic) interaction, an atom-to-face (carbonyl-aromatic) interaction, or no interaction at all. These interaction geometries are analyzed with regard to van der Waals, electrostatic, and possible charge-transfer effects. For the aromatic-aromatic interactions investigated in this study, with aromatic ring quadrupoles specifically "tuned" by the degree and pattern of fluorination, the structural results suggest that London forces and charge-transfer complexation dominate over weakly polar electrostatic interactions in the association of aromatic ring pairs.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/química , Flúor/química , Hidrocarburos Aromáticos/química , Unión Competitiva , Cristalografía por Rayos X , Humanos , Unión Proteica , Estructura Cuaternaria de ProteínaRESUMEN
Overexpression of the zinc enzyme carbonic anhydrase (CA; EC ) XII is observed in certain human cancers. This bitopic membrane protein contains an N-terminal extracellular catalytic domain, a membrane-spanning alpha-helix, and a small intracellular C-terminal domain. We have determined the three-dimensional structure of the extracellular catalytic domain of human CA XII by x-ray crystallographic methods at 1.55-A resolution. The structure reveals a prototypical CA fold; however, two CA XII domains associate to form an isologous dimer, an observation that is confirmed by studies of the enzyme in solution. The identification of signature GXXXG and GXXXS motifs in the transmembrane sequence that facilitate helix-helix association is additionally consistent with dimeric architecture. The dimer interface is situated so that the active site clefts of each monomer are clearly exposed on one face of the dimer, and the C termini are located together on the opposite face of the dimer to facilitate membrane interaction. The amino acid composition of the active-site cleft closely resembles that of the other CA isozymes in the immediate vicinity of the catalytic zinc ion, but differs in the region of the nearby alpha-helical "130's segment." The structure of the CA XII-acetazolamide complex is also reported at 1.50-A resolution, and prospects for the design of CA XII-specific inhibitors of possible chemotherapeutic value are discussed.
Asunto(s)
Anhidrasas Carbónicas/química , Proteínas de la Membrana/química , Proteínas de Neoplasias/química , Acetazolamida/química , Acetazolamida/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Catálisis , Cristalografía por Rayos X , Dimerización , Diseño de Fármacos , Humanos , Ratones , Datos de Secuencia Molecular , Conformación Proteica , Ratas , Proteínas Recombinantes de Fusión/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Zinc/químicaRESUMEN
The boronic acid-based arginine analogue S-(2-boronoethyl)-L-cysteine (BEC) has been synthesized and assayed as a slow-binding competitive inhibitor of the binuclear manganese metalloenzyme arginase. Kinetic measurements indicate a K(I) value of 0.4-0.6 microM, which is in reasonable agreement with the dissociation constant of 2.22 microM measured by isothermal titration calorimetry. The X-ray crystal structure of the arginase-BEC complex has been determined at 2.3 A resolution from crystals perfectly twinned by hemihedry. The structure of the complex reveals that the boronic acid moiety undergoes nucleophilic attack by metal-bridging hydroxide ion to yield a tetrahedral boronate anion that bridges the binuclear manganese cluster, thereby mimicking the tetrahedral intermediate (and its flanking transition states) in the arginine hydrolysis reaction. Accordingly, the binding mode of BEC is consistent with the structure-based mechanism proposed for arginase as outlined in Cox et al. [Cox, J. D., Cama, E., Colleluori D. M., Pethe, S., Boucher, J. S., Mansuy, D., Ash, D. E., and Christianson, D. W. (2001) Biochemistry 40, 2689-2701.]. Since BEC does not inhibit nitric oxide synthase, BEC serves as a valuable reagent to probe the physiological relationship between arginase and nitric oxide (NO) synthase in regulating the NO-dependent smooth muscle relaxation in human penile corpus cavernosum tissue that is required for erection. Consequently, we demonstrate that arginase is present in human penile corpus cavernosum tissue, and that the arginase inhibitor BEC causes significant enhancement of NO-dependent smooth muscle relaxation in this tissue. Therefore, human penile arginase is a potential target for the treatment of sexual dysfunction in the male.
Asunto(s)
Arginasa/antagonistas & inhibidores , Arginina/análogos & derivados , Arginina/metabolismo , Ácidos Borónicos/metabolismo , Inhibidores Enzimáticos/metabolismo , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Erección Peniana/fisiología , Animales , Arginasa/biosíntesis , Arginasa/genética , Arginasa/metabolismo , Arginina/farmacología , Unión Competitiva , Ácidos Borónicos/síntesis química , Ácidos Borónicos/farmacología , Calorimetría , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Cinética , Sustancias Macromoleculares , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Erección Peniana/efectos de los fármacos , Pene/irrigación sanguínea , Pene/enzimología , Pene/inervación , ARN Mensajero/biosíntesis , Conejos , Ratas , TermodinámicaRESUMEN
Arginase is a binuclear Mn(2+) metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. X-ray crystal structures of arginase complexed to substrate analogues N(omega)-hydroxy-L-arginine and N(omega)-hydroxy-nor-L-arginine, as well as the products L-ornithine and urea, complete a set of structural "snapshots" along the reaction coordinate of arginase catalysis when interpreted along with the X-ray crystal structure of the arginase-transition-state analogue complex described in Kim et al. [Kim, N. N., Cox, J. D., Baggio, R. F., Emig, F. A., Mistry, S., Harper, S. L., Speicher, D. W., Morris, Jr., S. M., Ash, D. E., Traish, A. M., and Christianson, D. W. (2001) Biochemistry 40, 2678-2688]. Taken together, these structures render important insight on the structural determinants of tight binding inhibitors. Furthermore, we demonstrate for the first time the structural mechanistic link between arginase and NO synthase through their respective complexes with N(omega)-hydroxy-L-arginine. That N(omega)-hydroxy-L-arginine is a catalytic intermediate for NO synthase and an inhibitor of arginase reflects the reciprocal metabolic relationship between these two critical enzymes of L-arginine catabolism.
Asunto(s)
Arginasa/química , Arginasa/metabolismo , Arginina/análogos & derivados , Sustitución de Aminoácidos/genética , Animales , Arginasa/antagonistas & inhibidores , Arginasa/genética , Arginina/química , Arginina/metabolismo , Unión Competitiva/genética , Catálisis , Cristalografía por Rayos X , Cisteína/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Histidina/genética , Sustancias Macromoleculares , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Ornitina/química , Ornitina/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato/genética , Urea/química , Urea/metabolismoRESUMEN
The structure of the trimeric, manganese metalloenzyme, rat liver arginase, has been previously determined at 2.1-A resolution (Kanyo, Z. F., Scolnick, L. R., Ash, D. E., and Christianson, D. W., (1996) Nature 383, 554-557). A key feature of this structure is a novel S-shaped oligomerization motif at the carboxyl terminus of the protein that mediates approximately 54% of the intermonomer contacts. Arg-308, located within this oligomerization motif, nucleates a series of intramonomer and intermonomer salt links. In contrast to the trimeric wild-type enzyme, the R308A, R308E, and R308K variants of arginase exist as monomeric species, as determined by gel filtration and analytical ultracentrifugation, indicating that mutation of Arg-308 shifts the equilibrium for trimer dissociation by at least a factor of 10(5). These monomeric arginase variants are catalytically active, with k(cat)/K(m) values that are 13-17% of the value for wild-type enzyme. The arginase variants are characterized by decreased temperature stability relative to the wild-type enzyme. Differential scanning calorimetry shows that the midpoint temperature for unfolding of the Arg-308 variants is in the range of 63.6-65.5 degrees C, while the corresponding value for the wild-type enzyme is 70 degrees C. The three-dimensional structure of the R308K variant has been determined at 3-A resolution. At the high protein concentrations utilized in the crystallizations, this variant exists as a trimer, but weakened salt link interactions are observed for Lys-308.
Asunto(s)
Arginasa/química , Arginasa/genética , Mutación , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Arginina/química , Rastreo Diferencial de Calorimetría , Catálisis , Cromatografía en Gel , Dicroismo Circular , Cristalografía por Rayos X , Dimerización , Espectroscopía de Resonancia por Spin del Electrón , Electrones , Cinética , Hígado/enzimología , Manganeso/química , Ratones , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Ratas , Temperatura , Ultracentrifugación , XenopusRESUMEN
Aromatic residues in the hydrophobic core of human carbonic anhydrase II (CAII) influence metal ion binding in the active site. Residues F93, F95, and W97 are contained in a beta-strand that also contains two zinc ligands, H94 and H96. The aromatic amino acids contribute to the high zinc affinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitution of these aromatic amino acids with smaller side chains enhances Cu(2+) affinity while decreasing Co(2+) and Zn(2+) affinity [Hunt, J. A., Mahiuddin, A., & Fierke, C. A. (1999) Biochemistry 38, 9054-9062]. Here, X-ray crystal structures of zinc-bound F93I/F95M/W97V and F93S/F95L/W97M CAIIs reveal the introduction of new cavities in the hydrophobic core, compensatory movements of surrounding side chains, and the incorporation of buried water molecules; nevertheless, the enzyme maintains tetrahedral zinc coordination geometry. However, a conformational change of direct metal ligand H94 as well as indirect (i.e., "second-shell") ligand Q92 accompanies metal release in both F93I/F95M/W97V and F93S/F95L/W97M CAIIs, thereby eliminating preorientation of the histidine ligands with tetrahedral geometry in the apoenzyme. Only one cobalt-bound variant, F93I/F95M/W97V CAII, maintains tetrahedral metal coordination geometry; F93S/F95L/W97M CAII binds Co(2+) with trigonal bipyramidal coordination geometry due to the addition of azide anion to the metal coordination polyhedron. The copper-bound variants exhibit either square pyramidal or trigonal bipyramidal metal coordination geometry due to the addition of a second solvent molecule to the metal coordination polyhedron. The key finding of this work is that aromatic core residues serve as anchors that help to preorient direct and second-shell ligands to optimize zinc binding geometry and destabilize alternative geometries. These geometrical constraints are likely a main determinant of the enhanced zinc/copper specificity of CAII as compared to small molecule chelators.
Asunto(s)
Aminoácidos/química , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Metales Pesados/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Cobalto/metabolismo , Cobre/metabolismo , Cristalografía por Rayos X , Humanos , Leucina/química , Metionina/química , Fenilalanina/química , Serina/química , Relación Estructura-Actividad , Especificidad por Sustrato , Triptófano/química , Valina/química , Zinc/metabolismoRESUMEN
Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr(465) and Tyr(381) act as acid catalysts, activating the epoxide ring and facilitating the formation of a covalent intermediate between the epoxide and the enzyme. To explore the role of these two residues, mutant enzymes were produced and the mechanism of action was analyzed. Enzyme assays on a series of substrates confirm that both Tyr(465) and Tyr(381) are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr(465) and Tyr(381) decreases the rate of binding and the formation of an intermediate, suggesting that both tyrosines polarize the epoxide moiety to facilitate ring opening. These two tyrosines are, however, not implicated in the hydrolysis of the covalent intermediate. Sequence comparisons showed that Tyr(465) is conserved in microsomal EHs. The substitution of analogous Tyr(374) with phenylalanine in the human microsomal EH dramatically decreases the rate of hydrolysis of cis-stilbene oxide. These results suggest that these tyrosines perform a significant mechanistic role in the substrate activation by EHs.
Asunto(s)
Epóxido Hidrolasas/metabolismo , Compuestos Epoxi/metabolismo , Tirosina/metabolismo , Animales , Secuencia de Bases , Catálisis , Cartilla de ADN , Epóxido Hidrolasas/genética , Humanos , Cinética , Ratones , MutaciónRESUMEN
[figure: see text] Linear free energy relationships between binding affinity and hydrophobicity for a library of fluoroaromatic inhibitors of F131V carbonic anhydrase II (CA) implicate three modes of interaction. X-ray crystal structures suggest that F131 interacts with fluoroaromatic inhibitors, while P202, on the opposite side of the active site cleft, serves as the site of the hydrophobic contact in the case of the F131V mutant. 2-Fluorinated compounds bind more tightly, perhaps due to the field effect of the nearby fluorine on the acidity of the amide proton.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/metabolismo , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Inhibidores de Anhidrasa Carbónica/química , Cristalografía/métodos , Flúor/química , Transferencia Lineal de Energía , Modelos Moleculares , Mutación , Conformación ProteicaRESUMEN
The 2.5-A resolution crystal structure of recombinant aristolochene synthase from the blue cheese mold, Penicillium roqueforti, is the first of a fungal terpenoid cyclase. The structure of the enzyme reveals active site features that participate in the cyclization of the universal sesquiterpene cyclase substrate, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. Metal-triggered carbocation formation initiates the cyclization cascade, which proceeds through multiple complex intermediates to yield one exclusive structural and stereochemical isomer of aristolochene. Structural homology of this fungal cyclase with plant and bacterial terpenoid cyclases, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of terpene biosynthesis.
Asunto(s)
Proteínas Fúngicas/química , Isomerasas/química , Penicillium/enzimología , Sitios de Unión , Cristalografía por Rayos X , Electrones , Evolución Molecular , Isomerasas/aislamiento & purificación , Magnesio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
The structures of two alkylurea inhibitors complexed with murine soluble epoxide hydrolase have been determined by x-ray crystallographic methods. The alkyl substituents of each inhibitor make extensive hydrophobic contacts in the soluble epoxide hydrolase active site, and each urea carbonyl oxygen accepts hydrogen bonds from the phenolic hydroxyl groups of Tyr(381) and Tyr(465). These hydrogen bond interactions suggest that Tyr(381) and/or Tyr(465) are general acid catalysts that facilitate epoxide ring opening in the first step of the hydrolysis reaction; Tyr(465) is highly conserved among all epoxide hydrolases, and Tyr(381) is conserved among the soluble epoxide hydrolases. In one enzyme-inhibitor complex, the urea carbonyl oxygen additionally interacts with Gln(382). If a comparable interaction occurs in catalysis, then Gln(382) may provide electrostatic stabilization of partial negative charge on the epoxide oxygen. The carboxylate side chain of Asp(333) accepts a hydrogen bond from one of the urea NH groups in each enzyme-inhibitor complex. Because Asp(333) is the catalytic nucleophile, its interaction with the partial positive charge on the urea NH group mimics its approach toward the partial positive charge on the electrophilic carbon of an epoxide substrate. Accordingly, alkylurea inhibitors mimic features encountered in the reaction coordinate of epoxide ring opening, and a structure-based mechanism is proposed for leukotoxin epoxide hydrolysis.
Asunto(s)
Inhibidores Enzimáticos/farmacocinética , Epóxido Hidrolasas/química , Epóxido Hidrolasas/metabolismo , Tirosina , Urea/análogos & derivados , Urea/farmacocinética , Secuencia de Aminoácidos , Toxinas Bacterianas/química , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Epóxido Hidrolasas/antagonistas & inhibidores , Exotoxinas/farmacocinética , Enlace de Hidrógeno , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Conformación Proteica , Urea/química , Urea/farmacologíaAsunto(s)
Arginasa/química , Arginasa/metabolismo , Manganeso/metabolismo , Animales , Arginasa/antagonistas & inhibidores , Dominio Catalítico , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Cinética , Manganeso/química , Modelos Moleculares , Estructura Cuaternaria de Proteína , Especificidad por SustratoAsunto(s)
Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Mamíferos , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Zinc/metabolismoAsunto(s)
Arginasa/análisis , Aminocaproatos/farmacología , Animales , Arginasa/antagonistas & inhibidores , Arginasa/metabolismo , Compuestos de Boro/farmacología , Compuestos Cromogénicos , Inhibidores Enzimáticos/farmacología , Guanidinas , Cinética , Hígado/enzimología , Nitrobencenos , Ratas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismoRESUMEN
The crystal structure of the complex between the binuclear manganese metalloenzyme arginase and the boronic acid analog of L-arginine, 2(S)-amino-6-boronohexanoic acid (ABH), has been determined at 1.7 A resolution from a crystal perfectly twinned by hemihedry. ABH binds as the tetrahedral boronate anion, with one hydroxyl oxygen symmetrically bridging the binuclear manganese cluster and a second hydroxyl oxygen coordinating to Mn2+A. This binding mode mimics the transition state of a metal-activated hydroxide mechanism. This transition state structure differs from that occurring in NO biosynthesis, thereby explaining why ABH does not inhibit NO synthase. We also show that arginase activity is present in the penis. Accordingly, the tight binding and specificity of ABH allows us to probe the physiological role of arginase in modulating the NO-dependent smooth muscle relaxation required for erection. Strikingly, ABH causes significant enhancement of nonadrenergic, noncholinergic nerve-mediated relaxation of penile corpus cavernosum smooth muscle, suggesting that arginase inhibition sustains L-arginine concentrations for NO synthase activity. Therefore, human penile arginase is a potential target for therapeutic intervention in the treatment of erectile dysfunction.
Asunto(s)
Aminocaproatos/metabolismo , Arginasa/química , Arginasa/metabolismo , Compuestos de Boro/metabolismo , Inhibidores Enzimáticos/metabolismo , Erección Peniana/fisiología , Pene/enzimología , Pene/fisiología , Aminocaproatos/química , Aminocaproatos/farmacología , Animales , Arginasa/antagonistas & inhibidores , Arginina/química , Arginina/metabolismo , Compuestos de Boro/química , Compuestos de Boro/farmacología , Cristalización , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Hígado/enzimología , Masculino , Modelos Moleculares , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Erección Peniana/efectos de los fármacos , Pene/efectos de los fármacos , Pene/inervación , Conformación Proteica , Conejos , Ratas , Relación Estructura-ActividadRESUMEN
The crystal structure of recombinant murine liver cytosolic epoxide hydrolase (EC 3.3.2.3) has been determined at 2.8-A resolution. The binding of a nanomolar affinity inhibitor confirms the active site location in the C-terminal domain; this domain is similar to that of haloalkane dehalogenase and shares the alpha/beta hydrolase fold. A structure-based mechanism is proposed that illuminates the unique chemical strategy for the activation of endogenous and man-made epoxide substrates for hydrolysis and detoxification. Surprisingly, a vestigial active site is found in the N-terminal domain similar to that of another enzyme of halocarbon metabolism, haloacid dehalogenase. Although the vestigial active site does not participate in epoxide hydrolysis, the vestigial domain plays a critical structural role by stabilizing the dimer in a distinctive domain-swapped architecture. Given the genetic and structural relationships among these enzymes of xenobiotic metabolism, a structure-based evolutionary sequence is postulated.
Asunto(s)
Carcinógenos/farmacocinética , Epóxido Hidrolasas/química , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/farmacocinética , Inactivación Metabólica , Hígado/enzimología , Mutágenos/farmacocinética , Animales , Cristalografía por Rayos X , Dimerización , Hidrolasas/química , Hidrólisis , Ratones , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Xenobióticos/metabolismoRESUMEN
An increase in arginase activity has been associated with the pathophysiology of a number of conditions, including an impairment in nonadrenergic and noncholinergic (NANC) nerve-mediated relaxation of the gastrointestinal smooth muscle. An arginase inhibitor may rectify this condition. We compared the effects of a newly designed arginase inhibitor, 2(S)-amino-6-boronohexanoic acid (ABH), with the currently available N(omega)-hydroxy-L-arginine (L-HO-Arg), on the NANC nerve-mediated internal anal sphincter (IAS) smooth-muscle relaxation and the arginase activity in the IAS and other tissues. Arginase caused an attenuation of the IAS smooth-muscle relaxations by NANC nerve stimulation that was restored by the arginase inhibitors. L-HO-Arg but not ABH caused dose-dependent and complete reversal of N(omega)-nitro-L-arginine-suppressed IAS relaxation that was similar to that seen with L-arginine. Both ABH and L-HO-Arg caused an augmentation of NANC nerve-mediated relaxation of the IAS. In the IAS, ABH was found to be approximately 250 times more potent than L-HO-Arg in inhibiting the arginase activity. L-HO-Arg was found to be 10 to 18 times more potent in inhibiting the arginase activity in the liver than in nonhepatic tissues. We conclude that arginase plays a significant role in the regulation of nitric oxide synthase-mediated NANC relaxation in the IAS. The advent of new and selective arginase inhibitors may play a significant role in the discrimination of arginase isozymes and have important pathophysiological and therapeutic implications in gastrointestinal motility disorders.