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PubMedDate not supplied

[Characterization of Escherichia hermannii by electrophoresis of esterases, acid phosphatase and glutamate and malate dehydrogenases].

Esterases, acid phosphatase and glutamate and malate dehydrogenases of 11 strains of Escherichia hermannii were analysed by horizontal electrophoresis in polyacrylamide agarose gel. Seven esterase bands were defined by their range of activity on synthetic substrates and their sensitivity or resistance to di-isopropyl fluorophosphate. These bands were different in activity and in mobility from those produced by E. coli strains. On the basis of variations in mobility of glutamate and malate dehydrogenases and in the number and mobility of esterases, the strains were divided into 3 zymotypes.

Acid Phosphatase↗

PubMed2002

Synthesis and stability study of a modified phenylpropionic acid linker-based esterase-sensitive prodrug.

An esterase-sensitive amide prodrug 1 with a modified phenylpropionic acid linker was synthesized. The prodrug can be converted to the drug using isolated porcine esterase and human plasma. Paraoxon, an esterase inhibitor, can inhibit prodrug-to-drug conversion. The conversion of prodrug 1 was via phenol intermediate 9 followed by a lactonization reaction to give lactone 2 and the drug.

Amides↗

PubMed2003

New fluorinated derivatives as esterase inhibitors. Synthesis, hydration and crossed specificity studies.

A variety of new fluorinated chemicals have been prepared for the first time and tested as inhibitors of esterases, one of the main enzymes involved in pheromone catabolism, in two economically important pests, the Egyptian armyworm Spodoptera littoralis (SL) and the Mediterranean corn borer Sesamia nonagrioides (SN). Using the respective major component of the pheromone as substrate, the K(m) and V(max) of the antennal esterase of both insects resulted to be 5.66 x 10(-4) M and 8.47 x 10(-6) Mmin(-1) for SL and 1.61 x 10(-7) M and 1.25 x 10(-7) Mmin(-1) for SN, pointing out that SN esterase has a higher affinity for its corresponding substrate than SL. In general, the trifluoromethyl ketones (TFMKs) exhibited higher inhibitory potency than the corresponding difluoromethyl ketones (DFMKs) or difluoroaldehydes (DFAs). The compounds appeared to hydrate differently in aqueous solution, the extent of hydration following the order: alpha,alpha-DFMKs<alpha,alpha-difluoro-beta-thioalkylmethyl ketones<TFMKs<beta-thiotrifluoromethyl ketones<alpha,alpha-DFAs. No clear correlation has been found between the K(hyd) and the inhibitory potency and no specificity has been found when the chemicals were assayed on extracts of both insects.

Algorithms↗

PubMed1998

Coumarin-based prodrugs. Part 3: Structural effects on the release kinetics of esterase-sensitive prodrugs of amines.

To study the structural effects on the release kinetics of a coumarin-based esterase-sensitive prodrug system, two series of compounds with varying structural features of the ester 'trigger' part and the amine 'drug' part were synthesized. The half-lives of the nine model prodrugs in the presence of porcine liver esterase ranged from about 2 min to 190 min. The steric bulkiness of the acyl group seems to have only a very minor effect on the half-lives of the esterase-triggered release of amines from the model prodrugs. The rate of the lactonization depends on the steric and electronic properties of the amine moiety.

Amines↗

PubMed1995

Lipases and esterases: a review of their sequences, structure and evolution.

This chapter aims to provide a brief review on the enzyme family of lipases and esterases. The sequences, 3D structures and pH dependent electrostatic signatures are presented and analyzed. Since the family comprises more than 100 sequences, we have tried to focus on the most interesting features from our perspective, which translates into finding similarities and differences between members of this family, in particular in and around the active sites, and to identify residues that are partially or totally conserved. Such residues we believe are either important for maintaining the structural scaf-fold of the protein or to maintain activity or specificity. The structure function relationship for these proteins is therefore of central interest. Can we uniquely identify a protein from this large family of sequences--and if so, what is the identifier? The protein family displays some highly complex features: many of the proteins are interfacially activated, i.e. they need to be in physical contact with the aggregated substrate. Access to the active site is blocked with either a loop fragment or an alpha-helical fragment in the absence of interfacial contact. Although the number of known, relevant protein 3D structures is growing steadily, we are nevertheless faced with a virtual explosion in the number of known or deduced amino acid sequences. It is therefore unrealistic to expect that all protein sequences within the foreseeable future will have their 3D structure determined by X-ray diffractional analysis or through other methods. When feasible the gene and/or the amino acid sequences will be analyzed from an evolutionary perspective. As the 3D folds are often remarkably similar, both among the triglyceride lipases as well as among the esterases, the functional diversities (e.g. specificity) must originate in differences in surface residue utilization, in particular of charged residues. The pH variations in the isopotential surfaces of some of the most interesting lipases are presented and a qualitative interpretation proposed. Finally we illustrate that NMR has potential for becoming an important tool in the study of lipases, esterases and their kinetics.

Amino Acid Sequence↗

PubMed1991

Cholesterol esterase catalyzed hydrolysis of mixed micellar thiophosphatidylcholines: a possible charge-relay mechanism.

Mechanistic features of cholesterol esterase catalyzed hydrolysis of two thiophospholipids, rac-1-(hexanoylthio)-2-hexanoyl-3-glycerophosphorylcholine (6TPC) and rac-1-(decanoylthio)-2-decano-yl-3-glycerophosphorylcholine (10TPC), have been characterized. The hydrolysis of 10TPC that is contained in mixed micelles with Triton X-100 occurs strictly at the micellar interface, since the reaction rate is independent of the micelle concentration but depends hyperbolically on the mole fraction of the substrate in the micelles. This latter observation allows one to calculate the interfacial kinetic parameters V*max and K*m. The hydrolyses of 10TPC and p-nitrophenyl butyrate are similarly inhibited by the transition state analogue inhibitor phenyl-n-butylborinic acid, and therefore, physiological and nonphysiological substrates are processed at the same active site. The similarity of k*cat values for the acyl-similar substrates 10TPC and p-nitrophenyl decanoate indicates that the phospholipase A1 activity of cholesterol esterase is partially rate limited by turnover of a decanoyl-enzyme intermediate. Solvent isotope effects on V*max and V*max/K*m (which monitors acylation only) are approximately 2-3 and are consistent with transition states that are stabilized by general acid-base proton transfers. Proton inventories of V*max/K*m indicate that simultaneous proton transfers stabilize the acylation transition state, which requires a multifunctional acid-base machinery (perhaps a charge-relay system) in the cholesterol esterase active site. Similar results are obtained for the 6TPC reaction, both in the presence and absence of Triton X-100 micelles.

Acetylcholinesterase↗

PubMed1976

Esterase activity of zinc neutral proteases.

The hydrolysis of a series of depsipeptides demonstrates that the zinc neutral endopeptidases of bacteria are active esterases. Esters such as BzGly-OPhe-Ala, BzGly-OLeu-Ala, and FA-Gly-OLeu-NH2 are hydrolyzed at rates three- to eightfold slower than are their exact peptide analogues, when hydrolyzed by thermolysin, Bacillus subtilis neutral protease and the neutral protease from Aeromonas proteolytica. Ester hydrolysis by zinc neutral proteases follows the characteristic preference for hydrophobic amino acids adjacent to the site of cleavage, discerned from the hydrolysis of peptide substrates. Removal of zinc from thermolysin abolishes the esterase activity of the native enzyme. Among the metals examined, only Co2+ and Zn2+ restore esterase activity to any significant extent, Co2+ restoring 50% and Zn2+ 100% of the native thermolysin activity. The hydrolysis of esters and peptides by thermolysin does not differ with respect to either the binding or catalytic steps. Substrate specificity, pH-rate profiles, inhibitor, and deuterium isotope effects are identical for both types of substrates.

Binding Sites↗

PubMed2004

Cholesterol crystal nucleation from enzymatically modified low-density lipoproteins: combined effect of sphingomyelinase and cholesterol esterase.

An assay detecting and quantifying cholesterol nucleation from low-density lipoproteins has been established. Förster resonance energy transfer between dehydroergosterol and dansylated lecithin becomes significantly alleviated as a consequence of conucleation of dehydroergosterol and cholesterol. The assay, in combination with dynamic light scattering, absorbance spectroscopy, and fluorescence microscopy, can be used to study aggregation and nucleation in model blood systems. Human plasma LDL was labeled with dehydroergosterol and dansylated lecithin by incubation with donor multilamellar liposomes and isolated by centrifugation. Exposure of labeled LDL (0.5 mg/mL of total lipids) to sphingomyelinase (0.0-0.2 unit/mL) led to modest particle aggregation but produced no changes in energy transfer and no crystallization. However, addition of sphingomyelinase produced significant particle aggregation, nucleation, and crystallization, in a dose-dependent fashion, in samples that were previously treated with the enzyme, cholesterol esterase (0.2 unit/mL). The combination of cholesterol esterase and sphingomyelinase led to a significant alleviation of energy transfer, which preceded by 24 h the appearance of fluorescent, microscopic sterol crystals. These results point to a synergistic effect between cholesterol esterase and sphingomyelinase, suggesting that mere aggregation of LDL is insufficient to promote nucleation, and crystal formation likely proceeds in the intracellular space after LDL uptake by macrophages.

Cholesterol↗

PubMed2006

Identification and characterization of O-acetylpeptidoglycan esterase: a novel enzyme discovered in Neisseria gonorrhoeae.

Modification of the bacterial cell wall heteropolymer peptidoglycan by addition of an acetyl group to the C-6 hydroxyl group of N-acetylmuramoyl residues is known to inhibit the activity of muramidases (lysozymes) of innate immune systems. The O-acetylation of peptidoglycan also precludes the action of intrinsic lytic transglycosylases, enzymes that require a free C-6 hydroxyl group to generate their 1,6-anhydromuropeptide products. This class of autolysins is ubiquitous in peptidoglycan-synthesizing bacteria as they are responsible for insertion of pores and flagella, spore formation, and the general metabolism of peptidoglycan. We recently discovered a cluster of genes in the Neisseria gonorrhoeae chromosome that are proposed to participate in peptidoglycan O-acetylation (Weadge, J. T., Pfeffer, J. M., and Clarke, A. J. (2005) BMC Microb. 5, 49). In the current study, we demonstrate that one of these genes, ape1 functions as an O-acetylpeptidoglycan esterase. The ape1 gene was cloned and overexpressed in Escherichia coli as a fusion protein with a hexa-histidine tag. The expressed protein was purified to apparent homogeneity and assayed for activity as an esterase using three different assays involving high-performance liquid chromatography and chromogenic detection methods which measured the release of ester-linked acetate from a variety of polymer and soluble substrates. These assays demonstrated that Ape1 has a higher specific activity on O-acetylated peptidoglycan compared to O-acetylated xylan. Consequently, Ape1 represents the first enzyme characterized as an O-acetylpeptidoglycan esterase. The physicochemical and kinetic parameters of Ape1 were determined using soluble chromogenic substrates for convenience. Thus, its pH optima for stability and activity were observed to be 6.0 and 6.2, respectively, while its optimum temperature for activity was 55 degrees C. Two forms of truncated Ape1 are generated in E. coli, one lacked the complete predicted N-terminal signal sequence, while the second involved a proteolytic cleavage within this signal sequence. The smaller truncated form was localized predominantly to the periplasm, whereas the larger form was mainly associated with the outer membrane, and to a lesser extent, the cytoplasmic membrane, sites expected for the maintenance of peptidoglycan.

Acetylation↗

PubMed1998

Structure of bovine pancreatic cholesterol esterase at 1.6 A: novel structural features involved in lipase activation.

The structure of pancreatic cholesterol esterase, an enzyme that hydrolyzes a wide variety of dietary lipids, mediates the absorption of cholesterol esters, and is dependent on bile salts for optimal activity, is determined to 1.6 A resolution. A full-length construct, mutated to eliminate two N-linked glycosylation sites (N187Q/N361Q), was expressed in HEK 293 cells. Enzymatic activity assays show that the purified, recombinant, mutant enzyme has activity identical to that of the native, glycosylated enzyme purified from bovine pancreas. The mutant enzyme is monomeric and exhibits improved homogeneity which aided in the growth of well-diffracting crystals. Crystals of the mutant enzyme grew in space group C2, with the following cell dimensions: a = 100.42 A, b = 54.25 A, c = 106.34 A, and beta = 104.12 degrees, with a monomer in the asymmetric unit. The high-resolution crystal structure of bovine pancreatic cholesterol esterase (Rcryst = 21.1%; Rfree = 25.0% to 1.6 A resolution) shows an alpha-beta hydrolase fold with an unusual active site environment around the catalytic triad. The hydrophobic C terminus of the protein is lodged in the active site, diverting the oxyanion hole away from the productive binding site and the catalytic Ser194. The amphipathic, helical lid found in other triglyceride lipases is truncated in the structure of cholesterol esterase and therefore is not a salient feature of activation of this lipase. These two structural features, along with the bile salt-dependent activity of the enzyme, implicate a new mode of lipase activation.

Acetylcholinesterase↗

PubMed2000

Tryptophan phosphorescence study of enzyme flexibility and unfolding in laboratory-evolved thermostable esterases.

Directed evolution of p-nitrobenzyl esterase (pNB E) has yielded eight generations of increasingly thermostable variants. The most stable esterase, 8G8, has 13 amino acid substitutions, a melting temperature 17 degrees C higher than the wild-type enzyme, and increased hydrolytic activity toward p-nitrophenyl acetate (pNPA), the substrate used for evolution, at all temperatures. Room-temperature activities of the evolved thermostable variants range from 3.5 times greater to 4.0 times less than wild type. The relationships between enzyme stability, catalytic activity, and flexibility for the esterases were investigated using tryptophan phosphorescence. We observed no correlation between catalytic activity and enzyme flexibility in the vicinity of the tryptophan (Trp) residues. Increases in stability, however, are often accompanied by decreases in flexibility, as measured by Trp phosphorescence. Phosphorescence data also suggest that the N- and C-terminal regions of pNB E unfold independently. The N-terminal region appears more thermolabile, yet most of the thermostabilizing mutations are located in the C-terminal region. Mutational studies show that the effects of the N-terminal mutations depend on one or more mutations in the C-terminal region. Thus, the pNB E mutants are stabilized by long-range, cooperative interactions between distant parts of the enzyme.

Directed Molecular Evolution↗

PubMed2002

Enantiomeric resolution of 2-aryl propionic esters with hyperthermophilic and mesophilic esterases: contrasting thermodynamic mechanisms.

The enantiomeric resolution of 2-aryl propionic esters by hyperthermophilic and mesophilic esterases was found to be governed by contrasting thermodynamic mechanisms. Entropic contributions predominated for mesophilic esterases from Candida rugosa and Rhizomucor miehei, while enthalpic forces controlled this resolution by the esterase from the extremely thermoacidophilic archaeon, Sulfolobus solfataricus P1. This disparity in thermodynamic mechanism can be attributed to the differences in conformational flexibility of mesophilic and thermophilic enzymes as they relate to the temperature range (4-70 degrees C) examined.

Candida↗

PubMed2004

Development of pyrethroid substrates for esterases associated with pyrethroid resistance in the tobacco budworm, Heliothis virescens (F.).

Assays to detect esterases associated with resistance to organophosphorus and pyrethroid insecticides in larvae of H. virescens were developed and evaluated. Cross-resistance to a variety of insecticides was measured in strains resulting from selection with either profenofos (OP-R) or cypermethrin (PYR-R), and resistance in both strains appeared to have a metabolic component. Esters were synthesized that coupled 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate, the acid moiety of some pyrethroid insecticides, with groups (e.g., p-nitrophenyl-) that could be detected spectrophotometrically following hydrolysis of the resulting esters. Activities toward these pyrethroid esters were significantly higher in both resistant strains than those in a susceptible reference strain. In addition, all pyrethroid esters significantly increased the toxicity of cypermethrin in bioassays with larvae from both PYR-R and OP-R strains. The biological and biochemical activities of these compounds are compared with those with more conventional esterase substrates and insecticide synergists, and the utility of pyrethroid esters as components of rapid assays for detecting esterases associated with insecticide resistance is discussed.

Animals↗

PubMed2006

Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112.

The gene encoding pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the estP gene revealed an open reading frame of 1914 bp encoding for a protein of 637 amino acid residues. No similarities were found by a database homology search using the nucleotide and deduced amino acid sequences of the esterases and lipases. EstP was heterologously expressed in E. coli and purified. The molecular mass of the native enzyme was approximately 73 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of EstP indicated molecular masses of 73 and 73.5 kDa, respectively, suggesting that EstP is a monomer. The purified EstP not only degraded many pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyzed rho-nitrophenyl esters of various fatty acids, indicating that EstP is an esterase with broad substrates. The K(m) for trans- and cis-permethrin and k(cat)/K(m) values indicate that EstP hydrolyzes both these substrates with higher efficiency than the carboxylesterases from resistant insects and mammals. The catalytic activity of EstP was strongly inhibited by Hg2+, Ag+, and rho-chloromercuribenzoate, whereas a less pronounced effect (3-8% inhibition) was observed in the presence of divalent cations, the chelating agent EDTA, and phenanthroline.

Amino Acid Sequence↗

PubMed2000

Esterase-sensitive nitric oxide donors of the diazeniumdiolate family: in vitro antileukemic activity.

We have designed a novel prodrug class that is stable in neutral aqueous media but releases bioactive nitric oxide (NO) on metabolism by esterase. Diazeniumdiolates of structure R(2)N-N(O)=N-OR', in which R' = Na, were reacted with BrCH(2)OAc to convert the spontaneously NO-releasing salts 1a (R(2)N = diethylamino) and 1b (R(2)N = pyrrolidino) to prodrugs 2a (AcOM-DEA/NO) and 2b (AcOM-PYRRO/NO), respectively, where R' = CH(2)OAc. In contrast to anions 1a and 1b (half-lives in pH 7.4 phosphate at 37 degrees C of 2 min and 3 s, respectively), 2a and 2b showed only minimal decomposition after 16 h under these conditions. Very rapid hydrolysis occurred in the presence of porcine liver esterase, however, with free anion 1a being observed as an intermediate in the esterase-induced generation of NO from 2a. The potential utility of this prodrug class is illustrated with a comparison of 1 and 2 as antiproliferative agents in NO-sensitive human leukemia cell lines HL-60 and U937. While the 72-h IC(50)'s for 1a and 1b (which generate NO throughout the medium) in HL-60 cell cultures were >600 microM, those of 2a and 2b were 8.3 and 6.4 microM, respectively. This result is consistent with our hypothesis that 2 is selectively hydrolyzed to 1 and thence to NO intracellularly. For U937 cells, the 72-h IC(50) for both 2a and 2b was 53 microM. By contrast, relatively high antiproliferative IC(50)'s (>100 microM in U937 cells) were observed for analogues in which R' = CH(2)CH(2)SC(O)Me, from which acetyl and 2-mercaptoethyl groups must be successively cleaved to free the NO-releasing diazeniumdiolate function. Within 24 h at initial concentrations of 50 microM, 2a and 2b induced apoptosis in 50% and 57% of the HL-60 cells, respectively (35% and 40% of the U937 cells, respectively). The data reveal significant in vitro antileukemic activity on the part of these novel compounds. Moreover, their substantial ease-of-handling advantages over the anionic diazeniumdiolates from which they are derived suggest their use as convenient agents for probing the biological roles of NO.

Animals↗

PubMed1998

An improved method for the simultaneous demonstration of mRNA and esterase activity at the human neuromuscular junction.

The aim of this study was to develop a simple means of studying the distribution of mRNA coding for post-synaptic proteins at the human neuromuscular junction. A reliable method by which to identify the junctions in tissue sections after in situ hybridization was essential. A method is described for combining the histochemical demonstration of esterase activity at the neuromuscular junction with autoradiographic localization of mRNA by in situ hybridization in the same cryostat section of skeletal muscle. The indigogenic esterase method of Strum and Hall-Craggs (1982) was modified in such a way that it is able to survive the multiple steps involved in in situ hybridization and autoradiography. The protocol is simple and reproducible and has been used successfully on sections of both rat and human skeletal muscle. To demonstrate the method, sections were reacted to reveal esterase activity and were then processed for in situ hybridization using a 35S-labelled probe specific for the epsilon-subunit of the acetylcholine receptor. The reaction product was retained after the lengthy in situ hybridization and autoradiographic procedures. To our knowledge, this is the first demonstration of acetylcholine receptor mRNA by in situ hybridization at human neuromuscular junctions.

Animals↗

PubMed1991

Amine prodrugs which utilize hydroxy amide lactonization. II. A potential esterase-sensitive amide prodrug.

In an effort to develop esterase-sensitive pro-prodrugs for amines, an amide derivative of 3-(2'-acetoxy-4',6'-dimethylphenyl)-3,3- dimethylpropionic acid (4-methoxyaniline amide 8) was synthesized and its stability investigated. This esterified hydroxy amide was found under all conditions to degrade via a two-step process initiated by acetyl ester hydrolysis generating the hydroxy amide intermediate 9a. The lactonization of this intermediate 9a in the second step resulted in the formation of 4-methoxyaniline (10) and 4,4,5,7-tetramethyl-3,4-dihydrocoumarin (1a). The pro-prodrug 8 was observed to possess the following half-lives at 37 degrees C under various conditions: 4030 min in phosphate buffer (50 mM, mu = 0.15) fixed to pH 7.4, 11.9 min in the same buffer containing a porcine liver esterase, 53.7 min in plasma, and 475 min in plasma containing diisopropylfluorophosphate. These results suggest that in a biological milieu the ester hydrolysis will occur by the enzymic hydrolysis rather than the chemical hydrolysis and that the enzymic hydrolysis of 8 in plasma is due, in part, to the action of serine-dependent esterases.

Amides↗

PubMed1998

A new esterase gene amplification involved in OP resistance in Culex pipiens mosquitoes from China.

Two overproduced esterases (A8 and B8) not previously described were found in southern China. They provide a low resistance level to organophosphate (OP) insecticides, and correspond to a coamplification of both esterase loci (Est-2 and Est-3) classically involved in OP resistance for this mosquito species. This coamplification is distinct from all other similar events thus far reported. The peculiar situation in southern China, where numerous OP resistance alleles at these two loci were found, is discussed in comparison with the Mediterranean situation, the only one with a similar diversity of overproduced esterases.

Animals↗