Asunto(s)
Simulación por Computador , Diseño de Fármacos , Proteínas/química , Proteínas/síntesis química , Técnicas Biosensibles , Proteínas de Unión al Calcio/síntesis química , Proteínas de Unión al Calcio/química , Catálisis , ADN/síntesis química , ADN/química , Esterasas/síntesis química , Esterasas/química , Esterasas/metabolismo , Gadolinio/química , Proteínas Fluorescentes Verdes/síntesis química , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Simulación de Dinámica Molecular , Nanopartículas/química , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/metabolismo , Ingeniería de Proteínas , Pliegue de Proteína , Proteínas/metabolismo , Proteínas/farmacología , Reproducibilidad de los Resultados , Virus Sincitiales Respiratorios/inmunología , Relación Estructura-Actividad , Vacunas Virales/química , Vacunas Virales/inmunología , Zinc/químicaRESUMEN
The accidental discovery of cisplatin some 50 years ago generated renewed interest in metallopharmaceuticals. Beyond cisplatin, many useful metallodrugs have been synthesized for the diagnosis and treatment of various diseases, but toxicity concerns, and the propensity to induce chemoresistance and secondary cancers make it imperative to search for novel metallodrugs that address these limitations. The Amino Terminal Cu(ii) and Ni(ii) (ATCUN) binding motif has emerged as a suitable template to design catalytic metallodrugs with nuclease and protease activities. Unlike their classical counterparts, ATCUN-based metallodrugs exhibit low toxicity, employ novel mechanisms to irreversibly inactivate disease-associated genes or proteins providing in principle, a channel to circumvent the rapid emergence of chemoresistance. The ATCUN motif thus presents novel strategies for the treatment of many diseases including cancers, HIV and infections caused by drug-resistant bacteria at the genetic level. This review discusses their design, mechanisms of action and potential for further development to expand their scope of application.
Asunto(s)
Antiinfecciosos/química , Antineoplásicos/química , Diseño de Fármacos , Compuestos Organometálicos/química , Antiinfecciosos/síntesis química , Antineoplásicos/síntesis química , Catálisis , Farmacorresistencia Microbiana , Esterasas/síntesis química , Esterasas/química , Humanos , Compuestos Organometálicos/síntesis química , Péptido Hidrolasas/síntesis química , Péptido Hidrolasas/químicaRESUMEN
Protein redesign methods aim to improve a desired property by carefully selecting mutations in relevant regions guided by protein structure. However, often protein structural requirements underlying biological characteristics are not well understood. Here, we introduce a methodology that learns relevant mutations from a set of proteins that have the desired property and demonstrate it by successfully improving production levels of two enzymes by Aspergillus niger, a relevant host organism for industrial enzyme production. We validated our method on two enzymes, an esterase and an inulinase, creating four redesigns with 5-45 mutations. Up to 10-fold increase in production was obtained with preserved enzyme activity for small numbers of mutations, whereas production levels and activities dropped for too aggressive redesigns. Our results demonstrate the feasibility of protein redesign by learning. Such an approach has great potential for improving production levels of many industrial enzymes and could potentially be employed for other design goals.
Asunto(s)
Aspergillus niger/enzimología , Evolución Molecular Dirigida/métodos , Esterasas/síntesis química , Proteínas Fúngicas/síntesis química , Glicósido Hidrolasas/síntesis química , Secuencia de Aminoácidos/genética , Aspergillus niger/genética , Clonación Molecular/métodos , Esterasas/genética , Proteínas Fúngicas/genética , Glicósido Hidrolasas/genética , Estructura Secundaria de ProteínaRESUMEN
Electrochemical biochips are an emerging tool for point-of-care diagnostic systems in medicine, food and environmental monitoring. In the current study, a thermostable reporter enzyme, esterase 2 (EST2) from Alicyclobacillus acidocaldarius, is used for specific and sensitive detection of bacteria by one-step rRNA/DNA hybridization between a bacterium-specific capture oligodeoxynucleotide (ODN), bacterial 16S rRNA and an uniform EST2-ODN reporter conjugate. The detection limit corresponds to approximately 500 colony forming units (cfu) Escherichia coli. Beside high sensitivity, the application of electrochemical biochips allows discrimination of two gram-negative and two gram-positive bacteria demonstrating the specificity and the potential for parallel detection of microorganisms. The feasibility of identification of foodborne bacteria was studied with meat juice contaminated with E. coli. This detection system has the capability to be applied for monitoring of bacterial food contamination.
Asunto(s)
Bacillus/genética , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Esterasas/metabolismo , Oligodesoxirribonucleótidos/genética , ARN Ribosómico 16S/análisis , Bacillus/enzimología , Proteínas Bacterianas/síntesis química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Técnicas Biosensibles/economía , Técnicas Biosensibles/instrumentación , Técnicas Electroquímicas/economía , Técnicas Electroquímicas/instrumentación , Esterasas/síntesis química , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/genética , Bacterias Grampositivas/enzimología , Bacterias Grampositivas/genética , Carne/análisis , Carne/microbiología , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico/métodos , Oligodesoxirribonucleótidos/síntesis química , ARN Bacteriano/análisis , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/aislamiento & purificación , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well as chemzymes that catalyze conjugate additions, cycloadditions, and self-replicating processes. The focus will be mainly on cyclodextrin-based chemzymes since they have shown to be good candidate structures to base an enzyme model skeleton on. In addition hereto, other molecules that encompass binding properties will also be presented.
Asunto(s)
Esterasas/síntesis química , Glicósido Hidrolasas/síntesis química , Oxidorreductasas/síntesis química , Sitios de Unión , Catálisis , Ciclodextrinas/síntesis química , Ciclodextrinas/química , Esterasas/química , Glicósido Hidrolasas/química , Cinética , Modelos Químicos , Oxidorreductasas/químicaRESUMEN
In this report, we present the first library of tripodal synthetic receptor molecules containing three different, temporarily N-terminal protected peptide arms capable of performing hydrolytic reactions. To construct this library, the orthogonally protected triazacyclophane (TAC)-scaffold was used in the preparation of a split-mix library of 19 683 resin bound tripodal receptor molecules. For the construction of the peptide arms, three different sets of amino acids were used, each focused on one part of the catalytic triad as found in several families of hydrolytic enzymes. Therefore, in the sets of amino acids used to assemble these tripeptides, basic (containing His and Lys), nucleophilic (containing Ser and Cys), or acidic (containing Asp and Glu) amino acid residues were present. In addition, nonfunctional hydrophobic amino acid residues were introduced. Possible unfavorable electrostatic interactions of charged N-termini or their acetylation during screening were circumvented by trifluoroacetylation of the N-terminal amines. Screening was performed with a known esterase substrate, 7-acetoxycoumarin, which upon hydrolysis gave the fluorescent 7-hydroxycoumarin, leading to fluorescence of beads containing a hydrolytically active synthetic receptor. Although many synthetic receptors contain catalytic triad combinations, apparently, only a few showed hydrolytic activity. Sequence analysis of the active receptors showed that carboxylate-containing amino acids are frequently found in the acidic arm and that substrate cleavage is mediated by lysine (noncatalytic) or histidine (catalytic) residues. Kinetic analysis of resynthesized receptors showed that catalysis depended on the number of histidine residues and was not assisted by significant substrate binding.
Asunto(s)
Esterasas/síntesis química , Imitación Molecular , Oligopéptidos/química , Secuencia de Aminoácidos , Sitios de Unión , Técnicas Químicas Combinatorias , Esterasas/química , Hidrólisis , Bibliotecas de Moléculas PequeñasRESUMEN
This work describes the synthesis of a library of fluorescent and/or biotinylated alkylphosphonate inhibitors being reactive towards serine hydrolases, especially lipases and esterases. Fluorescent inhibitors can be used for sensitive and rapid detection of active proteins by gel electrophoresis. Biotinylated inhibitors are applicable for the enrichment and isolation of active enzymes. Functionality as well as the different detection methods of the synthesized inhibitors were successfully tested with an enzyme preparation, namely cholesterol esterase from porcine pancreas (ppCE). Moreover, a biotinylated inhibitor was employed to enrich ppCE on avidin beads. Hence, our set of phosphonate inhibitors can be used for the detection and/or isolation of active serine hydrolases.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Esterasas/síntesis química , Lipasa/síntesis química , Compuestos Organofosforados/síntesis química , Animales , Avidina/química , Fluorescencia , Indicadores y Reactivos , Microscopía Fluorescente , Estructura Molecular , Compuestos Organofosforados/química , Páncreas/química , Esterol Esterasa/química , PorcinosRESUMEN
A 65,536-member combinatorial library of peptide dendrimers was prepared by split-and-mix synthesis and screened on solid support for esterolytic activity in aqueous buffer using 8-butyryloxypyrene-1,3,6-trisulfonate (2) as a fluorogenic substrate. Active sequences were identified by analysis of fluorescent beads. The corresponding dendrimers were resynthesized by solid-phase synthesis, cleaved from the resin, and purified by preparative reverse-phase HPLC. The dendrimers showed the expected catalytic activity in aqueous buffer. Catalysis was studied against a pannel of fluorogenic 8-acyloxypyrene-1,3,6-trisulfonate substrates. The catalytic peptide dendrimers display enzyme-like kinetics in aqueous buffer with substrate binding in the range K(M) approximately 0.1 mM, catalytic rate constants k(cat) approximately 0.1 min(-1), and specific rate accelerations over background up to k(cat)/k(uncat) = 10,000.
Asunto(s)
Técnicas Químicas Combinatorias , Dendrímeros/síntesis química , Esterasas/síntesis química , Péptidos/síntesis química , Arilsulfonatos/química , Dendrímeros/química , Esterasas/química , Hidrólisis , Cinética , Péptidos/químicaRESUMEN
An efficient enzyme model exhibiting enantioselective esterase activity was prepared by using molecular imprinting techniques. The enantiomerically pure phosphonic monoesters 4 L and 5 L were synthesized as stable transition-state analogues. They were used as templates connected by stoichiometric noncovalent interactions to two equivalents of the amidinium binding site monomer 1. After polymerization and removal of the template, the polymers were efficient catalysts for the hydrolysis of certain nonactivated amino acid phenylesters (2 L, 2 D, 3 L, 3 D) depending on the template used. Imprinted catalyst IP4 (imprinted with 4 L) enhanced the hydrolysis of the corresponding substrate 2 L by a factor of 325 relative to that of a buffered solution. Relative to a control polymer containing the same functionalities, prepared without template 4 L, the enhancement was still about 80-fold, showing the highest imprinting effect up to now. In cross-selectivity experiments a strong substrate selectivity of higher than three was found despite small differences in the structure of the substrate and template. Plots of initial velocities of the hydrolysis versus substrate concentration showed typical Michaelis-Menten kinetics with saturation behavior. From these curves, the Michaelis constant K(M) and the catalytic constant k(cat) can be calculated. The enantioselectivity shown in these values is most interesting. The ratio of the catalytic efficiency k(cat)/K(M), between the hydrolysis of 2 L- and 2 D-substrate with IP4, is 1.65. This enantioselectivity derives from both selective binding of the substrate (K(M)L/K(M)D=0.82), and from selective formation of the transition state (k(cat)L/k(cat)D=1.36). Thus, these catalysts give good catalysis as well as high imprinting and substrate selectivity. Strong competitive inhibition is caused by the template used in imprinting. This behavior is also quite similar to the behavior of natural enzymes, for which these catalysts are good models.
Asunto(s)
Esterasas/síntesis química , Esterasas/metabolismo , Polímeros/síntesis química , Polímeros/metabolismo , Tampones (Química) , Catálisis , Esterasas/química , Hidrólisis , Cinética , Leucina/análogos & derivados , Leucina/metabolismo , Conformación Molecular , Estructura Molecular , Polímeros/química , Estereoisomerismo , Valina/análogos & derivados , Valina/metabolismoRESUMEN
An artificial phosphotriesterase (PTE) was constructed by co-polymerization of 4(5)-vinylimidazole-Zn(2+)-methacrylic acid cluster with a divinylbenzene polymer. Compared with the spontaneous hydrolysis, the resulting polymer catalyst caused 105-fold rate acceleration towards the hydrolysis of diethyl p-nitrophenyl phosphate (Paraoxon). The catalytic activity of the polymer catalyst could be enhanced for 30% using molecular imprinting technique and the molecularly-imprinted catalyst (MIC) showed a turnover rate of 7.4 x 10(-2) s(-1) towards the hydrolysis of Paraoxon. The MIC also hydrolyzed thiophosphates and phosphorothiolate triester pesticides. Construction of an amperometric sensor employing the MIC as catalyst achieved a detection limit of 0.1 mM Paraoxon.
Asunto(s)
Técnicas Biosensibles/instrumentación , Enzimas Inmovilizadas/síntesis química , Esterasas/síntesis química , Paraoxon/química , Plaguicidas/análisis , Adsorción , Arildialquilfosfatasa , Materiales Biomiméticos/síntesis química , Técnicas Biosensibles/métodos , Catálisis , Electroquímica/instrumentación , Electroquímica/métodos , Esterasas/química , Imidazoles/química , Metacrilatos/química , Plaguicidas/química , Control de Calidad , Compuestos de Vinilo/química , Zinc/químicaRESUMEN
The industrial application of enzymes that can withstand harsh conditions has greatly increased over the past decade. This is mainly a result of the discovery of novel enzymes from extremophilic microorganisms. Recent advances in the study of extremozymes point to the acceleration of this trend. In particular, enzymes from thermophilic organisms have found the most practical commercial use to date because of their overall inherent stability. This has also led to a greater understanding of stability factors involved in adaptation of these enzymes to their unusual environments.
Asunto(s)
Archaea/enzimología , Bacterias/enzimología , Biotecnología , Enzimas/metabolismo , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/fisiología , Carbohidratos/síntesis química , Catálisis , Endopeptidasas/síntesis química , Endopeptidasas/metabolismo , Estabilidad de Enzimas/fisiología , Esterasas/síntesis química , Esterasas/metabolismo , Glicósido Hidrolasas/síntesis química , Glicósido Hidrolasas/metabolismo , Concentración de Iones de Hidrógeno , Lipasa/síntesis química , Lipasa/metabolismo , Nitrilos/metabolismo , Ósmosis , TemperaturaRESUMEN
Using site directed mutagenesis combined with chemical modification, we have developed a general and versatile method for the glycosylation of proteins which is virtually unlimited in the scope of proteins and glycans that may be conjugated and in which the site of glycosylation and the nature of the introduced glycan can be carefully controlled. We have demonstrated the applicability of this method through the synthesis of a library of 48 glycosylated forms of the serine protease subtilisin Bacillus lentus (SBL) as single, pure species. As part of our ongoing program to tailor the activity of SBL for use in peptide synthesis, we have screened these enzymes for activity against the esterase substrate succinyl-Ala-Ala-Pro-Phe-S-benzyl. Gratifyingly, 22 enzymes displayed greater than wild type (WT) activity. Glycosylation at positions 62, in the S2 pocket, resulted in five glycosylated forms of SBL that were 1.3- to 1.9-fold more active than WT. At position 217, in the S1' pocket, all glycosylations increased kcat/KM up to a remarkable 8.4-fold greater than WT for the glucosylated enzyme L217C-S-beta-Glc(Ac)3. Furthermore, the ratio of amidase to esterase activity, (kcat/KM)esterase/(kcat/KM)amidase (E/A), is increased relative to wild type for all 48 glycosylated forms of SBL. Again, the most dramatic changes are observed at positions 62 and 217 and L217C-S-beta-Glc(Ac)3 has an E/A that is 17.2-fold greater than WT. The tailored specificity and high activity of this glycoform can be rationalized by molecular modeling analysis, which suggests that the carbohydrate moiety occupies the S1' leaving group pocket and enhances the rate of deacylation of the acyl-enzyme intermediate. These glycosylated enzymes are ideal candidates for use as catalysts in peptide synthesis as they have greatly increased (kcat,KM)esterase and severely reduced (kcat/KM)amidase and will favor the formation of the amide bond over hydrolysis.
Asunto(s)
Esterasas/metabolismo , Subtilisina/química , Acetilación , Amidohidrolasas/síntesis química , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Bacillus/enzimología , Sitios de Unión , Técnicas Químicas Combinatorias , Esterasas/síntesis química , Esterasas/química , Glicoproteínas/síntesis química , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Relación Estructura-Actividad , Subtilisina/metabolismoRESUMEN
Organophosphorus hydrolase (OPH) is a bacterial enzyme that hydrolyzes a broad variety of OP neurotoxins, including chemical warfare agents and many widely used pesticides. OPH has extremely high hydrolytic efficiency with different phosphotriester and phophothiolester pesticides (k(cat) = 50-15,000 s(-1)) as well as phosphorofluorates such as DFP and the chemical warfare agents sarin and soman (k(cat) = 50-11,000 s(-1)). In contrast, the enzyme has much lower catalytic capabilities for phosphonothioate neurotoxins such as acephate or the chemical warfare agent VX [O-ethyl S-(2-diisopropyl aminoethyl) methylphosphonothioate] (k(cat) = 0.3-20 s(-1)). Different metal-associated forms of the enzyme have demonstrated varying hydrolytic capabilities for each of the OP neurotoxins, and the activity of OPH (Co2+) is consistently higher than that of OPH (Zn2+) by five- to 20-fold. Protein engineering strategies have exploited these metal-induced catalytic differences, and other slight modifications to the opd gene have resulted in significant enhancement of the rates of detoxification of the thioate pesticides and chemical warfare agents. In order to develop practical applications of OPH, other experiments have focused on improvement of enzyme production, localization, stability, and shelf-life, as well as efficient catalysis of substrates of interest.
Asunto(s)
Diseño de Fármacos , Esterasas/síntesis química , Esterasas/metabolismo , Animales , Arildialquilfosfatasa , Esterasas/genética , Esterasas/farmacología , Humanos , Hidrólisis , Ingeniería de Proteínas/métodos , Especificidad por SustratoRESUMEN
The synthetic peptide Chymohelizyme-1 (CHZ-1) exhibits esterase activity against carbobenzoxytyrosine p-nitrophenyl ester (ZTONP), carbobenzoxyalanine p-nitrophenyl ester (ZAONP), and t-butyloxy-carbonyltyrosine p-nitrophenyl ester (BocTONP). However, earlier reports of catalytic activity against less labile esters and amides have proven to be incorrect. The major reason for the errors appears to have been the omission of certain controls in the previous work. Although the catalytic triad does not appear to be functioning as designed, the catalytic activity of CHZ-1 does depend on the integrity of its primary structure. The pH dependence of hydrolysis of ZTONP points to general-base catalysis, whereas a preference for hydrophobic substrates suggest that the structure of CHZ-1 is performing some other role in assisting catalysis.
Asunto(s)
Quimotripsina/metabolismo , Esterasas/metabolismo , Péptidos/metabolismo , Alanina/análogos & derivados , Alanina/metabolismo , Alcohol Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Catálisis , Cromatografía Líquida de Alta Presión , Quimotripsina/síntesis química , Esterasas/síntesis química , Hidrólisis , Datos de Secuencia Molecular , Péptidos/síntesis química , Especificidad por Sustrato , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
A peptide having enzyme-like catalytic activity has been designed and synthesized. Computer modeling was used to design a bundle of four short parallel amphipathic helical peptides bearing the serine protease catalytic site residues serine, histidine, and aspartic acid at the amino end of the bundle in the same spatial arrangement as in chymotrypsin (ChTr). The necessary "oxyanion hole" and substrate binding pocket for acetyltyrosine ethyl ester, a classical ChTr substrate, were included in the design. The four chains were linked covalently at their carboxyl ends. The peptide has affinity for ChTr ester substrates similar to that of ChTr and hydrolyzes them at rates approximately 0.01 that of ChTr; total turnovers greater than 100 have been observed. The peptide is inhibited by ChTr specific inhibitors and is inactive toward benzoyl arginine ethyl ester, a trypsin substrate. The peptide is inactivated by heating above 60 degrees C, but recovers full catalytic activity upon cooling and lyophilization from acetic acid.
Asunto(s)
Quimotripsina/síntesis química , Esterasas/síntesis química , Péptidos/síntesis química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Quimotripsina/antagonistas & inhibidores , Quimotripsina/metabolismo , Simulación por Computador , Esterasas/antagonistas & inhibidores , Esterasas/metabolismo , Calor , Concentración de Iones de Hidrógeno , Hidrólisis , Modelos Químicos , Datos de Secuencia Molecular , Péptidos/antagonistas & inhibidores , Péptidos/metabolismo , Conformación Proteica , Especificidad por Sustrato , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Poly(His-Asp-Ser-Gly) was synthesized from the fully protected tetrapeptide active ester hydrochloride, which was prepared by stepwise coupling, using pentachlorophenyl ester and mixed anhydride methods. Complete deprotection of the protected tetrapeptide polymer was achieved by using 90% trifluoroacetic acid. The free polymer was dialyzed for 24 hr using a membrane (which retains molecules with molecular weights greater than 5000). The catalytic activity was determined by studying the hydrolysis of p-nitrophenyl acetate in 0.2 M phosphate buffer (pH 7.4) at 37 degrees. The catalytic coefficient of the dialyzed polymer was found to be 138 liters/mole/min.