RESUMEN
The discovery of new glycoside hydrolases that can be utilized in the chemoenzymatic synthesis of carbohydrates has emerged as a promising approach for various biotechnological processes. In this study, recombinant Ps_Cel5A from Pseudomonas stutzeri A1501, a novel member of the GH5_5 subfamily, was expressed, purified and crystallized. Preliminary experiments confirmed the ability of Ps_Cel5A to catalyze transglycosylation with cellotriose as a substrate. The crystal structure revealed several structural determinants in and around the positive subsites, providing a molecular basis for a better understanding of the mechanisms that promote and favour synthesis rather than hydrolysis. In the positive subsites, two nonconserved positively charged residues (Arg178 and Lys216) were found to interact with cellobiose. This adaptation has also been reported for transglycosylating ß-mannanases of the GH5_7 subfamily.
Asunto(s)
Proteínas Bacterianas/química , Celulasa/química , Celulosa/química , Pseudomonas stutzeri/enzimología , Triosas/química , Celulosa/metabolismo , Cristalización , Cristalografía por Rayos X/métodos , Escherichia coli , Glicosilación , Especificidad por Sustrato , Triosas/metabolismoRESUMEN
Phosphoenol pyruvate is the highest-energy phosphate found in living organisms and is one of the most versatile molecules in metabolism. Consequently, it is an essential intermediate in a wide variety of biochemical pathways, including carbon fixation, the shikimate pathway, substrate-level phosphorylation, gluconeogenesis and glycolysis. Triose glycolysis (generation of ATP from glyceraldehyde 3-phosphate via phosphoenol pyruvate) is among the most central and highly conserved pathways in metabolism. Here, we demonstrate the efficient and robust synthesis of phosphoenol pyruvate from prebiotic nucleotide precursors, glycolaldehyde and glyceraldehyde. Furthermore, phosphoenol pyruvate is derived within an α-phosphorylation controlled reaction network that gives access to glyceric acid 2-phosphate, glyceric acid 3-phosphate, phosphoserine and pyruvate. Our results demonstrate that the key components of a core metabolic pathway central to energy transduction and amino acid, sugar, nucleotide and lipid biosyntheses can be reconstituted in high yield under mild, prebiotically plausible conditions.
Asunto(s)
Evolución Química , Glucólisis , Origen de la Vida , Fosfoenolpiruvato/química , Triosas/química , Estructura Molecular , FosforilaciónAsunto(s)
Proteínas de Cloroplastos/metabolismo , Oryza/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Almidón/biosíntesis , Proteínas de Cloroplastos/genética , Oryza/genética , Proteínas de Transporte de Fosfato/genética , Fosfatos/química , Hojas de la Planta/metabolismo , Triosas/químicaRESUMEN
The direct conversion of biomass-derived 1,3-dihydroxyacetone (DHA) and formaldehyde to α-hydroxy-γ-butyrolactone (HBL) was achieved through the use of tin(iv) chloride and a small amount of water and the yield reached up to 70%. The reaction mechanism was also investigated by incorporating d2-formaldehyde into the reaction mixtures.
Asunto(s)
4-Butirolactona/química , Biomasa , Formaldehído/química , Compuestos de Estaño/química , Triosas/química , CatálisisRESUMEN
Rickettsia prowazekii is an obligate intracytosolic pathogen and the causative agent of epidemic typhus fever in humans. As an evolutionary model of intracellular pathogenesis, rickettsiae are notorious for their use of transport systems that parasitize eukaryotic host cell biochemical pathways. Rickettsial transport systems for substrates found only in eukaryotic cell cytoplasm are uncommon among free-living microorganisms and often possess distinctive mechanisms. We previously reported that R. prowazekii acquires triose phosphates for phospholipid biosynthesis via the coordinated activities of a novel dihydroxyacetone phosphate transport system and an sn-glycerol-3-phosphate dehydrogenase (K. M. Frohlich et al., J. Bacteriol. 192:4281-4288, 2010). In the present study, we have determined that R. prowazekii utilizes a second, independent triose phosphate acquisition pathway whereby sn-glycerol-3-phosphate is directly transported and incorporated into phospholipids. Herein we describe the sn-glycerol-3-phosphate and dihydroxyacetone phosphate transport systems in isolated R. prowazekii with respect to kinetics, energy coupling, transport mechanisms, and substrate specificity. These data suggest the existence of multiple rickettsial triose phosphate transport systems. Furthermore, the R. prowazekii dihydroxyacetone phosphate transport systems displayed unexpected mechanistic properties compared to well-characterized triose phosphate transport systems from plant plastids. Questions regarding possible roles for dual-substrate acquisition pathways as metabolic virulence factors in the context of a pathogen undergoing reductive evolution are discussed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Fosfatos/metabolismo , Rickettsia prowazekii/metabolismo , Triosas/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico Activo/fisiología , Fosfatos/química , Rickettsia prowazekii/genética , Especificidad por Sustrato , Triosas/químicaRESUMEN
Dietary fructose can benefit or hinder glycemic control, depending on the quantity consumed, and these contrasting effects are reflected by alterations in postprandial hepatic glycogen synthesis. Recently, we showed that ²H enrichment of glycogen positions 5 and 2 from deuterated water (²H2O) informs direct and indirect pathway contributions to glycogenesis in naturally feeding rats. Inclusion of position 6(S) ²H enrichment data allows indirect pathway sources to be further resolved into triose phosphate and Krebs cycle precursors. This analysis was applied to six rats that had fed on standard chow (SC) and six rats that had fed on SC plus 35% sucrose in their drinking water (HS). After 2 wk, hepatic glycogenesis sources during overnight feeding were determined by ²H2O administration and postmortem analysis of glycogen ²H enrichment at the conclusion of the dark period. Net overnight hepatic glycogenesis was similar between SC and HS rodents. Whereas direct pathway contributions were similar (403 ± 71 µmol/g dry wt HS vs. 578 ± 76 µmol/g dry wt SC), triose phosphate contributions were significantly higher for HS compared with SC (382 ± 61 vs. 87 ± 24 µmol/g dry wt, P < 0.01) and Krebs cycle inputs lower for HS compared with SC (110 ± 9 vs. 197 ± 32 µmol/g dry wt, P < 0.05). Analysis of plasma glucose ²H enrichments at the end of the feeding period also revealed a significantly higher fractional contribution of triose phosphate to plasma glucose levels in HS vs. SC. Hence, the ²H enrichment distributions of hepatic glycogen and glucose from ²H2O inform the contribution of dietary fructose to hepatic glycogen and glucose synthesis.
Asunto(s)
Fructosa/metabolismo , Glucógeno Hepático/metabolismo , Algoritmos , Métodos Analíticos de la Preparación de la Muestra , Animales , Glucemia/análisis , Agua Corporal/química , Ciclo del Ácido Cítrico , Óxido de Deuterio/metabolismo , Sacarosa en la Dieta/administración & dosificación , Fructosa/sangre , Glucosa/análogos & derivados , Glucosa/análisis , Glucosa/química , Cinética , Hígado/metabolismo , Glucógeno Hepático/química , Masculino , Resonancia Magnética Nuclear Biomolecular , Periodo Posprandial , Distribución Aleatoria , Ratas , Ratas Wistar , Triosas/química , Triosas/metabolismoRESUMEN
Efforts to improve the activity of cellulases, which catalyze the hydrolysis of insoluble cellulose, have been hindered by uncertainty surrounding the mechanistic origins of rate-limiting phenomena and by an incomplete understanding of complementary enzyme function. In particular, direct kinetic measurements of individual steps occurring after enzymes adsorb to the cellulose surface have proven to be experimentally elusive. This work describes an experimental and analytical approach, derived from a detailed mechanistic model of cellobiohydrolase action, for determining rates of initial- and processive-cut product generation by Trichoderma longibrachiatum cellobiohydrolase I (TlCel7A) as it catalyzes the hydrolysis of bacterial microcrystalline cellulose (BMCC) alone and in the presence of Talaromyces emersonii endoglucanase II (TemGH5). This analysis revealed that the rate of TlCel7A-catalyzed hydrolysis of crystalline cellulose is limited by the rate of enzyme complexation with glycan chains, which is shown to be equivalent to the rate of initial-cut product generation. This rate is enhanced in the presence of endoglucanase enzymes. The results confirm recent reports about the role of morphological obstacles in enzyme processivity and also provide the first direct evidence that processive length may be increased by the presence of companion enzymes, including small amounts of TemGH5. The findings of this work indicate that efforts to improve cellobiohydrolase activity should focus on enhancing the enzyme's ability to complex with cellulose chains, and the analysis employed provides a new technique for investigating the mechanism by which companion enzymes influence cellobiohydrolase activity.
Asunto(s)
Celulasa/química , Celulosa 1,4-beta-Celobiosidasa/química , Proteínas Fúngicas/química , Celulasa/genética , Celulasa/metabolismo , Celulosa/química , Celulosa 1,4-beta-Celobiosidasa/genética , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Oligosacáridos/química , Procesamiento Proteico-Postraduccional/genética , Especificidad por Sustrato/genética , Talaromyces/enzimología , Talaromyces/genética , Trichoderma/enzimología , Trichoderma/genética , Trichoderma/metabolismo , Triosas/químicaRESUMEN
Cellotriose and cellotetraose analogues carrying cyclohexene rings were developed as molecular probes which are expected to mimic the transition state conformation of hydrolysis by cellulases. The cyclohexene ring was placed at the pyranose ring being expected to locate the -1 subsite of the enzyme. In order to evaluate these probes, sulfur derivatives of cellotriose and cellotetraose were also synthesized as the enzyme tolerant analogues which mimic the stable conformations of the natural cellulose. The binding assays using differential scanning calorimetry revealed that introduction of the cyclohexene ring is effective to the complexation with an endoglucanase, NCE5 from Humicola insolens.
Asunto(s)
Celulasas/química , Celulosa/análogos & derivados , Tetrosas/síntesis química , Triosas/síntesis química , Rastreo Diferencial de Calorimetría , Celulasas/metabolismo , Celulosa/síntesis química , Celulosa/química , Ciclohexenos/química , Hidrólisis , Espectroscopía de Resonancia Magnética , Tetrosas/química , Triosas/químicaRESUMEN
We created 12 mutant enzymes (E11L, F40I, Y42L, N44L, N44Q, E47I, L62G, K64A, K64M, R137M, R137Q, and N139A) from the truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (TF-glucanase). The enzymes were used to investigate the structural and catalytic roles of specific amino acid residues located at the catalytic pocket and having direct interactions with glucose subsites of the product beta-1,3-1,4-cellotriose (CLTR). Fluorescence spectrometry showed no discernible changes in secondary structures among purified TF-glucanase and the mutants. Kinetic analyses showed E11L, F40I, Y42L, R137M, and R137Q with a >10-fold decrease of specific activity (11.2- to 67.4-fold), and E11L, N44Q, E47I, K64M, R137M, R137Q, and N139A with a 2.17- to 4.3-fold increase of K(m) value when compared with TF-glucanase. Notably, E11L, R137Q, R137M, F40I, and N139A showed the most significant decrease in catalytic efficiency relative to TF-glucanase, by 2155-, 84.9-, 48.5-, 41.1-, and 19.1-fold, respectively; the five mutants showed the greatest changes in comparative energy DeltaDeltaG(b), with values of 1.94 to 4.92 kcal/mol. Combined with results from kinetic and structure modeling analyses of all mutant enzymes and X-ray crystallography of F40I, we elucidate that Glu11, Phe40, Arg137, and Asn139 play a crucial role in the catalysis of TF-glucanase owing to their local and direct interaction through hydrogen bonds or van der Waals stacking interaction by aromatic rings onto the glucose subsites -3, -2, and -1 of CLTR/substrate. The overall globular structures in the wild-type and mutant F40I enzymes do not differ.
Asunto(s)
Aminoácidos/química , Proteínas Bacterianas/química , Fibrobacter/enzimología , Glicósido Hidrolasas/química , Secuencia de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Biocatálisis , Celulosa/química , Celulosa/metabolismo , Cristalografía por Rayos X , Fibrobacter/genética , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Especificidad por Sustrato , Temperatura , Triosas/química , Triosas/metabolismoRESUMEN
The glycoside hydrolase family 9 cellulase (Cel9) from Clostridium phytofermentans has a multi-modular structure and is essential for cellulose hydrolysis. In order to facilitate production and purification of Cel9, recombinant Cel9 was functionally expressed in Escherichia coli. Cel9 exhibited maximum activity at pH 6.5 and 65 degrees C on carboxymethyl cellulose in a 10-min reaction period. The hydrolysis products on regenerated amorphous cellulose (RAC) were cellotetraose (a major product), cellotriose, cellobiose and glucose, and 71-80% of the reducing sugars produced by Cel9 were in soluble form, suggesting that Cel9 was a processive endoglucanase. The highest synergy between C. phytofermentans Cel9 and C. phytofermentans cellobiohydrolase Cel48 on Avicel was about 1.8 at a ratio of about 1:5. Cel9 alone was sufficient to solublize filter paper while Cel48 was not; however, it enhanced the solublization process along with Cel9 synergistically. This study provided useful information for understanding of the cellulose hydrolysis mechanism of this cellulolytic bacterium with potential industrial importance.
Asunto(s)
Clostridium/metabolismo , Glicósido Hidrolasas/química , Celobiosa/química , Celulosa/análogos & derivados , Celulosa/química , Escherichia coli/metabolismo , Filtración , Glucosa/química , Concentración de Iones de Hidrógeno , Hidrólisis , Microbiología Industrial/métodos , Plásmidos/metabolismo , Temperatura , Tetrosas/química , Triosas/químicaRESUMEN
The prebiotic possibilities for the synthesis of interstellar carbohydrates through a protic variant of the formose reaction under gas phase conditions were studied. Ab initio calculations were used to evaluate potential mechanisms. Based on considerations of barrier heights and temperature variations in the Interstellar Medium the plausibility of extended sugar synthesis will be discussed.
Asunto(s)
Carbohidratos/síntesis química , Evolución Química , Origen de la Vida , Carbohidratos/química , Modelos Moleculares , Estructura Molecular , Tetrosas/síntesis química , Tetrosas/química , Triosas/síntesis química , Triosas/químicaRESUMEN
Reaction of triose sugars with ammonia under anaerobic conditions yielded autocatalytic products. The autocatalytic behavior of the products was examined by measuring the effect of the crude triose-ammonia reaction product on the kinetics of a second identical triose-ammonia reaction. The reaction product showed autocatalytic activity by increasing both the rate of disappearance of triose and the rate of formation of pyruvaldehyde, the product of triose dehydration. This synthetic process is considered a reasonable model of origin-of-life chemistry because it uses plausible prebiotic substrates, and resembles modern biosynthesis by employing the energized carbon groups of sugars to drive the synthesis of autocatalytic molecules.
Asunto(s)
Amoníaco/química , Piruvaldehído/química , Triosas/química , Carbono/química , Catálisis , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Iones , Reacción de Maillard , Modelos Químicos , Origen de la Vida , Compuestos de Amonio Cuaternario/química , Acetato de Sodio/química , Temperatura , Factores de TiempoRESUMEN
The galactosyl donor, 4,6-di-O-acetyl-2,3-di-O-benzyl-D-galactopyranosyl trichloroacetimidate, was efficiently coupled with regioselectively benzylated lactoside acceptors under standard conditions to stereoselectively afford the corresponding globotrioside and isoglobotrioside derivatives in very good yields. These glycosides were smoothly functionalized with a 6-(p-cinnamoylphenoxy)-hexyl tether tag as novel electrophilic thiol-specific carbohydrate reagents. Immobilization of the globotrioside conjugate to Thiopropyl Sepharose 6B for purification of B-subunit of Shiga toxin (StxB) and coupling of a model cysteine-containing protein (StxB-Z(n)-Cys) to the isoglobotrioside conjugate were both performed with high efficiency.
Asunto(s)
Oligosacáridos/síntesis química , Reactivos de Sulfhidrilo/síntesis química , Triosas/síntesis química , Secuencia de Carbohidratos , Chalcona/química , Cinamatos/química , Datos de Secuencia Molecular , Estructura Molecular , Oligosacáridos/química , Fenoles/química , Sefarosa/análogos & derivados , Sefarosa/química , Toxina Shiga/química , Toxina Shiga/aislamiento & purificación , Reactivos de Sulfhidrilo/química , Triosas/químicaRESUMEN
The cold-active phosphoglycerate kinase from the Antarctic bacterium Pseudomonas sp. TACII18 exhibits two distinct stability domains in the free, open conformation. It is shown that these stability domains do not match the structural N- and C-domains as the heat-stable domain corresponds to about 80 residues of the C-domain, including the nucleotide binding site, whereas the remaining of the protein contributes to the main heat-labile domain. This was demonstrated by spectroscopic and microcalorimetric analyses of the native enzyme, of its mutants, and of the isolated recombinant structural domains. It is proposed that the heat-stable domain provides a compact structure improving the binding affinity of the nucleotide, therefore increasing the catalytic efficiency at low temperatures. Upon substrate binding, the enzyme adopts a uniformly more stable closed conformation. Substrate-induced stability changes suggest that the free energy of ligand binding is converted into an increased conformational stability used to drive the hinge-bending motions and domain closure.
Asunto(s)
Fosfoglicerato Quinasa/química , Pseudomonas/enzimología , Aniones , Sitios de Unión , Calorimetría , Rastreo Diferencial de Calorimetría , Cationes , Cinética , Conformación Molecular , Mutagénesis , Mutación , Nucleótidos/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Espectrofotometría , Especificidad por Sustrato , Temperatura , Termodinámica , Factores de Tiempo , Triosas/química , Rayos UltravioletaRESUMEN
Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fsbeta-glucanase) catalyzes the specific hydrolysis of beta-1,4 glycosidic bonds adjacent to beta-1,3 linkages in beta-D-glucans or lichenan. This is the first report to elucidate the crystal structure of a truncated Fsbeta-glucanase (TFsbeta-glucanase) in complex with beta-1,3-1,4-cellotriose, a major product of the enzyme reaction. The crystal structures, at a resolution of 2.3 angstroms, reveal that the overall fold of TFsbeta-glucanase remains virtually unchanged upon sugar binding. The enzyme accommodates five glucose residues, forming a concave active cleft. The beta-1,3-1,4-cellotriose with subsites -3 to -1 bound to the active cleft of TFsbeta-glucanase with its reducing end subsite -1 close to the key catalytic residues Glu56 and Glu60. All three subsites of the beta-1,3-1,4-cellotriose adopted a relaxed C(1)4 conformation, with a beta-1,3 glycosidic linkage between subsites -2 and -1, and a beta-1,4 glycosidic linkage between subsites -3 and -2. On the basis of the enzyme-product complex structure observed in this study, a catalytic mechanism and substrate binding conformation of the active site of TFsbeta-glucanase is proposed.
Asunto(s)
Celulosa/metabolismo , Endo-1,3(4)-beta-Glucanasa/química , Endo-1,3(4)-beta-Glucanasa/metabolismo , Fibrobacter/enzimología , Oligosacáridos/química , Oligosacáridos/metabolismo , Triosas/metabolismo , Celulosa/química , Cristalografía por Rayos X , Endo-1,3(4)-beta-Glucanasa/genética , Fibrobacter/genética , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Especificidad por Sustrato , Triosas/químicaRESUMEN
Tin chlorides, SnCl2 and SnCl4.5H2O are excellent catalysts for the reactions of trioses, dihydroxyacetone and glyceraldehyde with alcohols (MeOH, EtOH and nBuOH) to give alkyl lactates, whose reaction mechanism involves the intermediary formation of pyruvic aldehyde followed by its esterification, which is distinctively promoted by tin halides.
Asunto(s)
Alcoholes/química , Lactatos/síntesis química , Compuestos de Estaño/química , Triosas/química , Catálisis , Lactatos/química , Estructura Molecular , SolucionesRESUMEN
The role of the highly reactive triose sugars glyceraldehyde and glyceraldehyde-3-phosphate in protein cross-linking and other amino acid modifications during the Maillard reaction was investigated. From the incubation of glyceraldehyde with N (alpha)-acetyl-L-lysine and N (alpha)-acetyl-L-arginine, we isolated four new Maillard reaction pyridinium compounds named 'triosidines'. Two of them, 'lys-hydroxy-triosidine' [1-(5-amino-5-carboxypentyl)-3-[(5-amino-5-carboxypentylamino)methyl]-5-hydroxypyridinium] and 'arg-hydroxy-triosidine' [2-(4-amino-4-carboxybutylamino)-8-(5-amino-5-carboxypentyl)-6-hydroxy-3,4-dihydro-pyrido[2,3-d]pyrimidin-8-ium] are fluorescent, UV-active Lys-Lys and Lys-Arg cross-links respectively. Their structures were identified by NMR and MS. In addition, two UV-active lysine adducts, 'trihydroxy-triosidine' [1-(5-amino-5-carboxypentyl)-3,4-dihydroxy-5-(hydroxymethyl)pyridinium] and 'triosidine carbaldehyde' [1-(5-amino-5-carboxypentyl)-3-formylpyridinium] were tentatively identified by MS. All structures involve six sugar-derived carbons as part of the heterocyclic ring. Of the two novel cross-links, only arg-hydroxy-triosidine was formed by glyceraldehyde-3-phosphate, an intermediate metabolite of the glycolytic pathway. Lys-hydroxy-triosidine and arg-hydroxy-triosidine were detected in human and porcine corneas treated with glyceraldehyde. The HPLC-fluorescence identification was confirmed by MS. Triosidines were also formed from dihydroxyacetone, a widely used artificial sun-tanning agent. Triosidines are expected to be useful tools in tissue engineering, where the utilization of highly reactive sugars is needed to stabilize the loose matrix. In addition, they are expected to be present in selected biological conditions, such as on consumption of a high fructose diet, and syndromes associated with high glyceraldehyde excretion, such as Fanconi Syndrome, fructose-1,6-diphosphatase deficiency and tyrosinaemia.
Asunto(s)
Aminoácidos/química , Arginina/química , Lisina/química , Compuestos de Piridinio/química , Triosas/química , Arginina/análogos & derivados , Reactivos de Enlaces Cruzados , Gliceraldehído/química , Lisina/análogos & derivados , Espectroscopía de Resonancia Magnética , Reacción de Maillard , Espectrometría de Masas , Estructura Molecular , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
The crystal structure of the Clostridium cellulovorans carbohydrate-binding module (CBM) belonging to family 17 has been solved to 1.7 A resolution by multiple anomalous dispersion methods. CBM17 binds to non-crystalline cellulose and soluble beta-1,4-glucans, with a minimal binding requirement of cellotriose and optimal affinity for cellohexaose. The crystal structure of CBM17 complexed with cellotetraose solved at 2.0 A resolution revealed that binding occurs in a cleft on the surface of the molecule involving two tryptophan residues and several charged amino acids. Thermodynamic binding studies and alanine scanning mutagenesis in combination with the cellotetraose complex structure allowed the mapping of the CBM17 binding cleft. In contrast to the binding groove characteristic of family 4 CBMs, family 17 CBMs appear to have a very shallow binding cleft that may be more accessible to cellulose chains in non-crystalline cellulose than the deeper binding clefts of family 4 CBMs. The structural differences in these two modules may reflect non-overlapping binding niches on cellulose surfaces.
Asunto(s)
Celulasa/química , Celulasa/metabolismo , Celulosa/análogos & derivados , Celulosa/química , Celulosa/metabolismo , Clostridium/química , Mutación/genética , Oligosacáridos/metabolismo , Tetrosas/química , Tetrosas/metabolismo , Alanina/genética , Alanina/metabolismo , Sitios de Unión , Calorimetría , Celulasa/clasificación , Celulasa/genética , Clostridium/enzimología , Clostridium/genética , Cristalografía por Rayos X , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Oligosacáridos/química , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Electricidad Estática , Especificidad por Sustrato , Termodinámica , Volumetría , Triosas/química , Triosas/metabolismo , Triptófano/metabolismoRESUMEN
The formation of pyruvaldehyde from triose sugars was catalyzed by poly-l-lysine contained in a small dialyzer with a 100 molecular weight cut off (100 MWCO) suspended in a much larger triose substrate reservoir at pH 5.5 and 40 degrees C. The polylysine confined in the dialyzer functioned as a catalytic flow reactor that constantly brought in triose from the substrate reservoir by diffusion to offset the drop in triose concentration within the reactor caused by its conversion to pyruvaldehyde. The catalytic polylysine solution (400 mM, 0.35 mL) within the dialyzer generated pyruvaldehyde with a synthetic intensity (rate/volume) that was 3400 times greater than that of the triose substrate solution (12 mM, 120 mL) outside the dialyzer. Under the given conditions the final yield of pyruvaldehyde was greater than twice the weight of the polylysine catalyst. During the reaction the polylysine catalyst was poisoned presumably by reaction of its amino groups with aldehyde reactants and products. Similar results were obtained using a dialyzer with a 500 MWCO. The dialyzer method of catalyst containment was selected because it provides a simple and easily manipulated experimental system for studying the dynamics and evolutionary development of confined autocatalytic processes related to the origin of life under anaerobic conditions.