RESUMEN
The moon's monthly cycle synchronizes reproduction in countless marine organisms. The mass-spawning bristle worm Platynereis dumerilii uses an endogenous monthly oscillator set by full moon to phase reproduction to specific days. But how do organisms recognize specific moon phases? We uncover that the light receptor L-Cryptochrome (L-Cry) discriminates between different moonlight durations, as well as between sun- and moonlight. A biochemical characterization of purified L-Cry protein, exposed to naturalistic sun- or moonlight, reveals the formation of distinct sun- and moonlight states characterized by different photoreduction- and recovery kinetics of L-Cry's co-factor Flavin Adenine Dinucleotide. In Platynereis, L-Cry's sun- versus moonlight states correlate with distinct subcellular localizations, indicating different signaling. In contrast, r-Opsin1, the most abundant ocular opsin, is not required for monthly oscillator entrainment. Our work reveals a photo-ecological concept for natural light interpretation involving a "valence interpreter" that provides entraining photoreceptor(s) with light source and moon phase information.
Asunto(s)
Criptocromos , Luna , Luz , Opsinas , Reproducción , Luz SolarRESUMEN
Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases.
Asunto(s)
Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Trypanosoma brucei rhodesiense/enzimología , Activación Enzimática , Concentración de Iones de Hidrógeno , Modelos Moleculares , Dominios ProteicosRESUMEN
A model system of two related enzymes with conserved binding sites, namely N-myristoyltransferase from two different organisms, was studied to decipher the driving forces that lead to selective inhibition in such cases. Using a combination of computational and experimental tools, two different selectivity-determining features were identified. For some ligands, a change in side-chain flexibility appears to be responsible for selective inhibition. Remarkably, this was observed for residues orienting their side chains away from the ligands. For other ligands, selectivity is caused by interfering with a water molecule that binds more strongly to the off-target than to the target. On the basis of this finding, a virtual screen for selective compounds was conducted, resulting in three hit compounds with the desired selectivity profile. This study delivers a guideline on how to assess selectivity-determining features in proteins with conserved binding sites and to translate this knowledge into the design of selective inhibitors.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/metabolismo , Diseño de Fármacos , Leishmania major/metabolismo , Modelos Moleculares , Aciltransferasas/química , Sitios de Unión/fisiología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Leishmania major/enzimología , LigandosRESUMEN
The Water-Soluble Chlorophyll Protein (WSCP) of Brassicaceae is a remarkably stable tetrapyrrole-binding protein that, by virtue of its simple design, is an exceptional model to investigate the interactions taking place between pigments and their protein scaffold and how they affect the photophysical properties and the functionality of the complexes. We investigated variants of WSCP from Lepidium virginicum (Lv) and Brassica oleracea (Bo), reconstituted with Chlorophyll (Chl) b, to determine the mechanisms by which the different Chl binding sites control their Chl a/b specificities. A combined Raman and crystallographic investigation has been employed, aimed to characterize in detail the hydrogen-bond network involving the formyl group of Chl b. The study revealed a variable degree of conformational freedom of the hydrogen bond networks among the WSCP variants, and an unexpected mixed presence of hydrogen-bonded and not hydrogen-bonded Chls b in the case of the L91P mutant of Lv WSCP. These findings helped to refine the description of the mechanisms underlying the different Chl a/b specificities of WSCP versions, highlighting the importance of the structural rigidity of the Chl binding site in the vicinity of the Chl b formyl group in granting a strong selectivity to binding sites.
Asunto(s)
Clorofila A/química , Clorofila/química , Enlace de Hidrógeno , Proteínas de Plantas/química , Brassicaceae/metabolismo , Cristalografía por Rayos X , Fotosíntesis , Conformación Proteica , Solubilidad , Espectrometría Raman , Agua/químicaRESUMEN
Water-soluble chlorophyll proteins (WSCP) from Brassicaceae form homotetrameric chlorophyll (Chl)-protein complexes binding one Chl per apoprotein and no carotenoids. Despite the lack of photoprotecting pigments, the complex-bound Chls displays a remarkable stability toward photodynamic damage. On the basis of a mutational study, we show that not only the presence of the phytyls is necessary for photoprotection in WSCPs, as we previously demonstrated, but also is their correct conformation and localization. The extreme heat stability of WSCP also depends on the presence of the phytyl chains, confirming their relevance for the unusual stability of WSCP.
RESUMEN
We altered the chlorophyll (Chl) binding sites in various versions of water-soluble chlorophyll protein (WSCP) by amino acid exchanges to alter their preferences for either Chl a or Chl b. WSCP is ideally suited for this mutational analysis since it forms a tetrameric complex with only four identical Chl binding sites. A loop of 4-6 amino acids is responsible for Chl a versus Chl b selectivity. We show that a single amino acid exchange within this loop changes the relative Chl a/b affinities by a factor of 40. We obtained crystal structures of this WSCP variant binding either Chl a or Chl b. The Chl binding sites in these structures were compared with those in the major light-harvesting complex (LHCII) of the photosynthetic apparatus in plants to search for similar structural features involved in Chl a/b binding specificity.
Asunto(s)
Clorofila A/metabolismo , Clorofila/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Brassica , Clorofila/química , Clorofila/genética , Clorofila A/química , Clorofila A/genética , Lepidium , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Agua/metabolismoRESUMEN
Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa ß-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/química , Acido Graso Sintasa Tipo II/química , Potasio/química , Pseudomonas aeruginosa/química , Proteínas Recombinantes de Fusión/química , Adamantano/química , Secuencias de Aminoácidos , Aminobenzoatos/química , Anilidas/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Benzamidas/química , Dominio Catalítico , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Descubrimiento de Drogas , Escherichia coli/genética , Escherichia coli/metabolismo , Acido Graso Sintasa Tipo II/antagonistas & inhibidores , Acido Graso Sintasa Tipo II/genética , Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Secundaria de Proteína , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/genética , Pseudomonas aeruginosa/enzimología , Proteínas Recombinantes de Fusión/genética , Salicilatos/químicaRESUMEN
Macromolecular oligomeric assemblies are involved in many biochemical processes of living organisms. The benefits of such assemblies in crowded cellular environments include increased reaction rates, efficient feedback regulation, cooperativity and protective functions. However, an atom-level structural determination of large assemblies is challenging due to the size of the complex and the difference in binding affinities of the involved proteins. In this study, we propose a novel combinatorial greedy algorithm for assembling large oligomeric complexes from information on the approximate position of interaction interfaces of pairs of monomers in the complex. Prior information on complex symmetry is not required but rather the symmetry is inferred during assembly. We implement an efficient geometric score, the transformation match score, that bypasses the model ranking problems of state-of-the-art scoring functions by scoring the similarity between the inferred dimers of the same monomer simultaneously with different binding partners in a (sub)complex with a set of pregenerated docking poses. We compiled a diverse benchmark set of 308 homo and heteromeric complexes containing 6 to 60 monomers. To explore the applicability of the method, we considered 48 sets of parameters and selected those three sets of parameters, for which the algorithm can correctly reconstruct the maximum number, namely 252 complexes (81.8%) in, at least one of the respective three runs. The crossvalidation coverage, that is, the mean fraction of correctly reconstructed benchmark complexes during crossvalidation, was 78.1%, which demonstrates the ability of the presented method to correctly reconstruct topology of a large variety of biological complexes.
Asunto(s)
Biología Computacional/métodos , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Modelos Moleculares , Proteínas/química , Proteínas/metabolismo , Algoritmos , Unión Proteica , Conformación Proteica , Programas InformáticosRESUMEN
The transport of sugars across the plasma membrane is a critical step in the utilization of glucose and fructose by Saccharomyces cerevisiae during must fermentations. Variations in the molecular structure of hexose transporters and kinases may affect the ability of wine yeast strains to finish sugar fermentation, even under stressful wine conditions. In this context, we sequenced and compared genes encoding the hexose transporter Hxt3p and the kinases Hxk1p/Hxk2p of Saccharomyces strains and interspecies hybrids with different industrial usages and regional backgrounds. The Hxt3p primary structure varied in a small set of amino acids, which characterized robust yeast strains used for the production of sparkling wine or to restart stuck fermentations. In addition, interspecies hybrid strains, previously isolated at the end of spontaneous fermentations, revealed a common amino acid signature. The location and potential influence of the amino acids exchanges is discussed by means of a first modelled Hxt3p structure. In comparison, hexokinase genes were more conserved in different Saccharomyces strains and hybrids. Thus, molecular variants of the hexose carrier Hxt3p, but not of kinases, correlate with different fermentation performances of yeast.
Asunto(s)
Alelos , Transferencia de Gen Horizontal , Hexoquinasa/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vino/microbiología , Secuencia de Bases , Quimera , Etanol/metabolismo , Fermentación , Fructosa/metabolismo , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Hexoquinasa/metabolismo , Conformación Proteica , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Polyphenoloxidases (PPO) of the type-3 copper protein family are considered to be catecholoxidases catalyzing the oxidation of o-diphenols to their corresponding quinones. PPO from Grenache grapes has recently been reported to display only diphenolase activity. In contrast, we have characterized PPOs from Dornfelder and Riesling grapes which display both monophenolase and diphenolase activity. Ultracentrifugation and size exclusion chromatography indicated that both PPOs occur as monomers with Mr of about 38kDa. Non-reducing SDS-PAGE shows two bands of about 38kDa exhibiting strong activity. Remarkably, three bands up to 60kDa displayed only very weak PPO activity, supporting the hypothesis that the C-terminal domain covers the entrance to the active site. Molecular dynamic analysis indicated that the hydroxyl group of monophenolic substrates can bind to CuA after the flexible but sterically hindering Phe 259 swings away on a picosecond time scale.
Asunto(s)
Catecol Oxidasa/química , Vitis/química , Vino/análisis , Catálisis , Cobre/análisis , Oxidación-ReducciónRESUMEN
Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (M(W) = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, ß = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.
Asunto(s)
Proteínas de Artrópodos/química , Hemocianinas/química , Multimerización de Proteína , Escorpiones , Animales , Cristalización , Cristalografía por Rayos X , Electrones , Modelos Moleculares , Estructura Cuaternaria de ProteínaRESUMEN
Hemocyanins are multimeric oxygen-transport proteins in the hemolymph of many arthropods and mollusks. The overall molecular architecture of arthropod and molluscan hemocyanin is very different, although they possess a similar binuclear type 3 copper center to bind oxygen in a side-on conformation. Gastropod hemocyanin is a 35 nm cylindrical didecamer (2 × 10-mer) based on a 400 kDa subunit. The latter is subdivided into eight paralogous "functional units" (FU-a to FU-h), each with an active site. FU-a to FU-f contribute to the cylinder wall, whereas FU-g and FU-h form the internal collar complex. Atomic structures of FU-e and FU-g, and a 9 Å cryoEM structure of the 8 MDa didecamer are available. Recently, the structure of keyhole limpet hemocyanin FU-h (KLH1-h) was presented as a C(α) -trace at 4 Å resolution. Unlike the other seven FU types, FU-h contains an additional C-terminal domain with a cupredoxin-like fold. Because of the resolution limit of 4 Å, in some loops, the course of the protein backbone could not be established with high certainty yet. Here, we present a refined atomic structure of FU-h (KLH1-h) obtained from low-resolution refinement, which unambiguously establishes the course of the polypeptide backbone and reveals the disulfide bridges as well as the orientation of bulky amino acids.
Asunto(s)
Disulfuros/química , Hemocianinas/química , Modelos MolecularesRESUMEN
BACKGROUND: Guinea pigs are considered to be genetically adapted to a high altitude environment based on the consistent finding of a high oxygen affinity of their blood. METHODOLOGY/PRINCIPAL FINDINGS: The crystal structure of guinea pig hemoglobin at 1.8 A resolution suggests that the increased oxygen affinity of guinea pig hemoglobin can be explained by two factors, namely a decreased stability of the T-state and an increased stability of the R2-state. The destabilization of the T-state can be related to the substitution of a highly conserved proline (P44) to histidine (H44) in the alpha-subunit, which causes a steric hindrance with H97 of the beta-subunit in the switch region. The stabilization of the R2-state is caused by two additional salt bridges at the beta1/beta2 interface. CONCLUSIONS/SIGNIFICANCE: Both factors together are supposed to serve to shift the equilibrium between the conformational states towards the high affinity relaxed states resulting in an increased oxygen affinity.
Asunto(s)
Adaptación Fisiológica , Altitud , Cobayas/fisiología , Hemoglobinas/química , Estructura Cuaternaria de Proteína , Animales , Cristalografía por Rayos X , Modelos Moleculares , Estabilidad ProteicaRESUMEN
We present a modular, collaborative, open-source architecture for rigid body modelling based upon small angle scattering data, named sas_rigid. It is designed to provide a fast and extensible scripting interface using the easy-to-learn Python programming language. Features include rigid body modelling to result in static structures and three-dimensional probability densities using two different algorithms.
Asunto(s)
Modelos Moleculares , Complejos Multiproteicos/química , Dispersión del Ángulo Pequeño , Método de Montecarlo , ProteínasRESUMEN
Allergens from cockroaches cause major asthma-related health problems worldwide. Among them Per a 3 belongs to the most potent allergens. Although the sequences of some members of the Per a 3-family are known, their biochemical and biophysical properties have not been investigated. Here we present for the first time a thorough structural characterization of these allergens, which have recently been tested to induce an increase of allergy specific indicators in blood of Europeans. We isolated two Per a 3 isoforms, which occur freely dissolved in the hemolymph as hexamers with molecular masses of 465+/-25kDa (P II) and 512+/-25kDa (P I). Their sedimentation coefficients (S(20,W)) were determined to be 17.4+/-0.7 S (P II) and 19.0+/-0.9 S (P I), respectively. Sequence analysis revealed that P II consists of two subunit types known as allergens Per a 3.01 and Per a 3.0201, while PI consists of a new allergenic subunit type designated as Per a 3.03. A 3D model of the hexameric allergen Per a 3 was obtained by homology modelling. Almost all of the recently predicted 11 putative antigenic peptides and reported IgE-epitopes could be located on the surface of the hexamer, thus being freely accessible in the hexameric structure of the native molecules. We propose this might contribute to their allergic potential as well as their extreme stability with respect to temperature.
Asunto(s)
Alérgenos/inmunología , Alérgenos/aislamiento & purificación , Proteínas de Insectos/inmunología , Proteínas de Insectos/aislamiento & purificación , Periplaneta/inmunología , Alérgenos/química , Alérgenos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Proteínas de Insectos/química , Proteínas de Insectos/genética , Espectrometría de Masas , Microscopía Electrónica , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Isoformas de Proteínas , Alineación de Secuencia , Análisis de Secuencia de ADN , UltracentrifugaciónRESUMEN
Haemocyanins are multimeric oxygen transport proteins, which bind oxygen to type 3 copper sites. Arthropod haemocyanins contain 75-kDa subunits, whereas molluscan haemocyanins contain 350-400-kDa subunits comprising seven or eight different 50 kDa FUs (functional units) designated FU-a to FU-h, each with an active site. FU-h possesses a tail of 100 amino acids not present in the other FUs. In the present study we show by X-ray crystallography that in FU-h of KLH1 (keyhole-limpet-haemocyanin isoform 1) the structure of the tail domain is cupredoxin-like but contains no copper. The copper-free domain 3 in arthropod haemocyanin subunits has also recently been reinterpreted as being cupredoxin-like. We propose that the cupredoxin-like domain in both haemocyanin types once served to upload copper to the active site of the oxygen-binding domain.
Asunto(s)
Gastrópodos/química , Hemocianinas/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Azurina/química , Sitios de Unión/genética , Cobre/química , Cristalografía por Rayos X , Gastrópodos/genética , Hemocianinas/clasificación , Hemocianinas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Filogenia , Homología de Secuencia de AminoácidoRESUMEN
In the Common woodlouse Porcellio scaber (Crustacea: Isopoda: Oniscidea), experimental immune challenge did not induce the expression of pro-phenoloxidase that, in most other invertebrates studied thus far, can be activated into phenoloxidase via an activation cascade upon immune challenge. Instead, Porcellio hemocyanin proved to exhibit catecholoxidase activity upon activation. However, none of the activating factors known from other invertebrates other than SDS-treatment resulted in activation of hemocyanin into a functional phenoloxidase in vitro. The distinct characteristics of isopod hemocyanin are reflected by the quaternary structure of the hemocyanin dodecamers that differs from that of other crustacean hemocyanins in that the two hexamers share a common 3-fold rotation axis and have an angular offset of 60 degrees against each other. Accordingly, the sequence of Porcellio hemocyanin can be distinguished clearly from other crustacean hemocyanins and in a phylogenetic analysis forms a cluster with other isopod and amphipod hemocyanins. We propose a peracarid-type hemocyanin that may have evolved in response to its required multiple functions in respiration and immune response, while phenoloxidase sensu strictu is lacking.
Asunto(s)
Hemocianinas/metabolismo , Sistema Inmunológico/inmunología , Isópodos/metabolismo , Monofenol Monooxigenasa/metabolismo , Animales , Catecol Oxidasa/metabolismo , Electroforesis , Precursores Enzimáticos/metabolismo , Expresión Génica , Hemocianinas/genética , Hemocianinas/ultraestructura , Hemocitos/química , Concentración de Iones de Hidrógeno , Sistema Inmunológico/efectos de los fármacos , Isópodos/genética , Isópodos/inmunología , Microscopía Electrónica , Oxidación-Reducción , Fenoles/metabolismo , Filogenia , Subunidades de Proteína/clasificación , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Dodecil Sulfato de Sodio/farmacología , Espectrometría de Fluorescencia , EspectrofotometríaRESUMEN
Hemoglobin is the versatile oxygen carrier in the blood of vertebrates and a key factor for adaptation to live in high altitudes. Several structural changes are known to account for increased oxygen affinity in hemoglobin of altitude adapted animals such as llama and barheaded goose. Guinea pigs are adapted to live in high altitudes in the Andes and consequently their hemoglobin has an increased oxygen affinity. However, the structural changes responsible for the adaptation of guinea pig hemoglobin are unknown. Here we report the crystallization of guinea pig hemoglobin in the presence of 2.6 M ammonium sulfate and a preliminary analysis of the crystals. Crystals diffract up to a resolution of 2.0 A. They are orthorhombic with space group C 2 2 2(1) and cell dimensions a = 84.08 A, b = 90.21 A and c = 83.44 A.
Asunto(s)
Aclimatación/fisiología , Altitud , Hemoglobinas/química , Animales , Cristalización , Cristalografía por Rayos X , CobayasRESUMEN
Phenoloxidases occur in almost all organisms, being essentially involved in various processes such as the immune response, wound healing, pigmentation and sclerotization in arthropods. Many hemocyanins are also capable of phenoloxidase activity after activation. Notably, in chelicerates, a phenoloxidase has not been identified in the hemolymph, and thus hemocyanin is assumed to be the physiological phenoloxidase in these animals. Although phenoloxidase activity has been shown for hemocyanin from several chelicerate species, a characterization of the enzymatic properties is still lacking. In this article, the enzymatic properties of activated hemocyanin from the tarantula Eurypelma californicum are reported, which was activated by SDS at concentrations above the critical micellar concentration. The activated state of Eurypelma hemocyanin is stable for several hours. Dopamine is a preferred substrate of activated hemocyanin. For dopamine, a K(M) value of 1.45 +/- 0.16 mm and strong substrate inhibition at high substrate concentrations were observed. Typical inhibitors of catecholoxidase, such as l-mimosine, kojic acid, tyramine, phenylthiourea and azide, also inhibit the phenoloxidase activity of activated hemocyanin. This indicates that the activated hemocyanin behaves as a normal phenoloxidase.
Asunto(s)
Catecol Oxidasa/química , Catecol Oxidasa/metabolismo , Hemocianinas/química , Hemocianinas/metabolismo , Arañas/enzimología , Animales , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Cinética , Dodecil Sulfato de Sodio/farmacología , Especificidad por SustratoRESUMEN
Phenoloxidases and hemocyanins have similar type 3 copper centers although they perform different functions. Hemocyanins are oxygen carriers, while phenoloxidases (tyrosinase/catecholoxidase) catalyze the initial step in melanin synthesis. Tyrosinases catalyze two subsequent reactions, whereas catecholoxidases catalyze only the second one. Recent results indicate that hemocyanins can also function as phenoloxidases and here we show for the first time that hemocyanin can be converted to phenoloxidase. Furthermore, its substrate specificity can be switched between catecholoxidase and tyrosinase activity depending on effectors such as hydroxymethyl-aminomethan (Tris) and Mg(2+)-ions. This demonstrates that substrate specificity is not caused by a chemical modification of the active site.