A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 degrees C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 degrees C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C8) with Km and kcat values of 71 microM and 14,700 s(-1), respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.     

A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification,characterization, and expression

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 °C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 °C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C_4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C₈) with K_m and k_cat values of 71 μM and 14,700 s⁻¹, respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.     

Paralogous gene analysis reveals a highly enantioselective 1,2-O-isopropylideneglycerol caprylate esterase of Bacillus subtilis.

Carboxylesterase NP of Bacillus subtilis Thai I-8, characterized in 1992 as a very enantioselective (S)-naproxen esterase, was found to show no enantiopreference towards (S)-1,2-O-isopropylideneglycerol (IPG) esters. The ybfK gene was identified by the B. subtilis genome project as an unknown gene with homology to carboxylesterase NP. The purpose of the present study was to characterize the ybfK gene product in order to determine whether this paralogue of carboxylesterase NP had an altered or enhanced stereospecificity. The ybfK gene was cloned and expressed in B. subtilis using a combination of two strong promoters in a multicopy vector. The enzyme was purified from the cytoplasm of B. subtilis by means of anion exchange and hydrophobic interaction chromatography. The purified YbfK is an enzyme of 296 amino acids and shows an apparent molecular mass of 32 kDa (SDS/PAGE). Comparison of the activities of YbfK and carboxylesterase NP towards caprylate esters of IPG revealed that YbfK produces (S)-IPG with 99.9% enantioselectivity. Therefore, we conclude that we have isolated a paralogue of carboxylesterase NP that can be used for the enantioselective production of (S)-IPG.     

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.     

Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans

Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40°C. The enzyme retained activity after incubation at pHs ranging from 8 to11 for 12 h at 37°C and 6 to 8 for 24 h at 37°C. It retained 80% of its activity after incubation at 30 to 70°C for 30 min and lost 50% of its activity after incubation for 15 min at 80°C. Noticeable activation of the enzyme is observed when Fe²⁺ ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu²⁺, Fe³⁺, Hg²⁺, and Zn²⁺ ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.     

A thermoactive uropygial esterase from chicken: purification, characterisation and synthesis of flavour esters

A lipolytic activity was located in the chicken uropygial glands, from which a carboxylesterase (CUE) was purified. Pure CUE has an apparent molecular mass of 50 kDa. The purified esterase displayed its maximal activity (200 U/mg) on short-chain triacylglycerols (tributyrin) at a temperature of 50°C. No significant lipolytic activity was found when medium chain (trioctanoin) or long chain (olive oil) triacylglycerols were used as substrates. The enzyme retained 75% of its maximal activity when incubated during 2h at 50°C. The NH(2)-terminal amino acid sequence showed similarities with the esterase purified recently from turkey pharyngeal tissue. Esterase activity remains stable after its incubation during 30 min in presence of organic solvents such as hexane or butanol. CUE is a serine enzyme since it was inactivated by phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor. The purified enzyme, which tolerates the presence of some organic solvent and a high temperature, can be used in non-aqueous synthesis reactions. Hence, the uropygial esterase immobilised onto CaCO(3) was tested to produce the isoamyl and the butyl acetate (flavour esters). Reactions were performed at 50°C in presence of hexane. High synthesis yields of 91 and 67.8% were obtained for isoamyl and butyl acetate, respectively.     

Expression of carboxylesterase and lipase genes in rat liver cell-types

Approximately 80% of the body vitamin A is stored in liver stellate cells with in the lipid droplets as retinyl esters. In low vitamin A status or after liver injury, stellate cells are depleted of the stored retinyl esters by their hydrolysis to retinol. However, the identity of retinyl ester hydrolase(s) expressed in stellate cells is unknown. The expression of carboxylesterase and lipase genes in purified liver cell-types was investigated by real-time PCR. We found that six carboxylesterase and hepatic lipase genes were expressed in hepatocytes. Adipose triglyceride lipase was expressed in Kupffer cells, stellate cells and endothelial cells. Lipoprotein lipase expression was detected in Kupffer cells and stellate cells. As a function of stellate cell activation, expression of adipose triglyceride lipase decreased by twofold and lipoprotein lipase increased by 32-fold suggesting that it may play a role in retinol ester hydrolysis during stellate cell activation.     

Extracellular carboxylesterase activity of Fusarium graminearum

Summary The extracellular carboxylesterase produced by Fusarium graminearum was investigated. The esterase showed optimum activity at 37°C and at pH 8.7–9.0. Increased activity was observed when whey as a growth substrate was supplemented with either Tween 40 or olive oil. Good activity was also obtained when the fungus was grown on a semi-solid bran culture medium. The esterase showed increased activity towards esters containing short-chain fatty acids.     

Newly isolated Streptomyces spp. as enantioselective biocatalysts: hydrolysis of 1,2-O-isopropylidene glycerol racemic esters

AIMS: To identify microbial strains with esterase activity able to enantioselectively hydrolyse esters of (R,S)-1,2-O-isopropylidene glycerol. METHODS AND RESULTS: The microbial hydrolysis of various racemic esters of 1,2-O-isopropylidene glycerol (IPG) was attempted by screening among Streptomyces spp. previously selected on the basis of their carboxylesterase activity. The best results were observed in the hydrolysis of butyrate ester and two strains appeared promising as they showed opposite enantioselectivity: Streptomyces sp. 90852 gave predominantly (S)-IPG, while strain 90930 mostly gave the R-alcohol. Streptomyces sp. 90930 was identified as Streptomyces violaceusniger, whereas Streptomyces sp. 90852 is a new species belonging to the Streptomyces violaceus taxon. The carboxylesterase belonging to strain 90852 gave a maximum value of enantiomeric ratio (E) of 14-16. This strain was lyophilized and used as dry mycelium for catalysing the synthesis of isopropylidene glycerol butyrate in heptane showing reaction rate and enantioselectivity (E = 6.6) lower than what observed for the hydrolysis. CONCLUSIONS: A new esterase with enantioselective activity towards (R,S)-IPG butyrate has been selected. The best enantioselectivity is similar or even better than the highest reported value in the literature with commercial enzymes. The enzyme is produced by a new species belonging to the S. violaceus taxon. SIGNIFICANCE AND IMPACT OF THE STUDY: New esterases from streptomycetes can be employed for the enantioselective hydrolysis of chiral esters derived from primary alcohols, not efficiently resolved with commercial enzymes.     

四种类型杀虫剂对麦长管蚜的温度效应及其与主要解毒酶的关系

为了明确杀虫剂毒力受温度的影响及其程度,本文测定了4大类8种药剂在10～25℃下对麦长管蚜的毒力;并测定了麦长管蚜Sitobion avenae(Fabricius)在不同温度下2个解毒酶和1个靶标酶的活性差异。结果表明,高效氯氰菊酯对麦长管蚜表现不规则负温度系数,啶虫脒表现不规则正温度系数,高效氟氯氰菊酯对麦长管蚜的毒力不受温度影响,其他药剂(辛硫磷、毒死蜱、灭多威、丁硫克百威、吡虫啉)均表现为明显的正温度系数效应,以有机磷类杀虫剂表现最为明显,毒死蜱温度系数高达57.70。酶活性实验表明:麦长管蚜在高温下GST活性增强,羧酸酯酶和乙酰胆碱酯酶活性降低。它们的变化规律表明:GST活性与负温度系数密切相关,正温度系数与羧酸酯酶活性和靶标酶乙酰胆碱酯酶活性有关。     

Purification and properties of an acetyl specific carboxylesterase from Nocardia mediterranei

Summary An acetyl specific carboxylesterase has been purified from Nocardia mediterranei. The purified enzyme is homogeneous as shown by SDS polyacrylamide gel electrophoresis. The esterase has a molecular weight of 68,000 and is composed of two identical subunits. The enzyme exhibits optimal activity at pH 7.5 and at 35°C and is stable below 40°C. The enzyme activity is inhibited by several sulfhydryl reagents. The esterase hydrolyzes preferentially acetyl esters. Propionyl esters are cleaved very slowly whereas butyryl esters are no substrates at all. In addition, the esterase shows a pronounced regiospecificity. On the other hand the enantiospecificity is rather low as demonstrated by the hydrolysis of prochiral and racemic substrates.     

A comparative study of the effect of vinyl phosphoric acid esters on cholinesterase and carboxylesterase activities in mammals and arthropods

Studies have been made of the effect of organophosphorus inhibitors on cholinesterase and carboxylesterase from various mammals (human erythrocytes, mouse brain, blood serum of mouse and rat, blood serum of horse) and arthropods (Calliphora vicina, Schizaphis graminum, Myzus persicae, Sitophilus oryzae, Pseudococcus maritimus, Tetranychus urticae). Organophosphorus inhibitors were presented by esters of vynylphosphoric acid containing normal and branched alkyls in the phosphoryl part of the molecule. The increase of the radical up to a propyl one increased the effect of organophosphorus inhibitors with respect to cholinesterase from the majority of the arthropods investigated. Organophosphorus compound with an isopropyl radical was found to be weaker for all the enzymes studied. Extremely high sensitivity of carboxylesterase from all arthropods to all organophosphorus inhibitors was noted; in some of the cases, anticarboxylesterase activity of all drugs was 2-3 orders higher than anticholinesterase one (P. maritimus, T. urticae). Regularities established for cholinesterase practically completely were confirmed on carboxylesterase. This finding evidently reveals similar structure of catalytic surface at the vicinity of esterase center in both enzymes.     

Comparison and functional characterisation of three homologous intracellular carboxylesterases of Bacillus subtilis

Enzymatic hydrolysis of racemic mixtures may provide an attractive method for the enantiopure production of chiral pharmaceuticals. For example, the carboxylesterase NP of Bacillus subtilis Thai I-8 is an excellent biocatalyst in the kinetic resolution of NSAID esters, such as naproxen and ibuprofen methyl esters. Two homologues of this enzyme were identified when the genome sequence of B. subtilis 168 was revealed in 1997. We characterised one of the homologous, YbfK, as a very enantioselective 1,2-O-isopropylidene-sn-glycerol caprylate esterase, while only modest enantioselectivity towards the naproxen ester was observed. The other homologue, the carboxylesterase NA has not been characterised yet. The purpose of the present study was to fully characterise these three highly homologous esterases with respect to their applicability towards the enantiospecific hydrolysis of a wide range of compounds. The esterase genes were cloned and expressed in B. subtilis using a combination of two strong promotors in a multi-copy vector. After purification of the enzymes from the cytoplasm of B. subtilis, the biochemical and enantioselective properties of the enzymes were determined. Although all carboxylesterases have similar physico-chemical properties, comparison of their specific activities and enantioselectivities towards several compounds revealed rather different substrate specificities. We conclude that carboxylesterase NP and carboxylesterase NA are particularly suited for the enzymatic conversion of naproxen esters, while YbfK offers enantiopure (+)-IPG from its caprylate ester. Given the carboxylesterase activities of the esterases it has been proposed to rename the nap gene of B. subtilis 168 into cesA and the ybfK gene into cesB.     

Purification and properties of a highly enantioselective l-menthyl acetate hydrolase from Burkholderia cepacia

A highly enantioselective l-menthyl acetate esterase was purified to homogeneity from Burkholderia cepacia ATCC 25416, with a recovery of 4.8% and a fold purification of 22.7. The molecular weight of the esterase was found to be 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was “MGARTDA”, and there was no homology in contrast to other Burkholderia sp. esterases. This enzyme preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity and high hydrolytic activity, while long-chain l-menthyl esters were poor substrates. Considered its substrate specificity and N-terminal sequence, this esterase was concluded as a new enzyme belonging to the carboxylesterase group (EC 3.1.1.1) of esterase family. The optimum temperature and pH for enzyme activity using racemic menthyl acetate as substrate were 30 °C and 7.0, respectively. The esterase was more stable in the pH range of 7.0–9.0 and temperature range of 30–40 °C. Hydrolytic activity was enhanced by Ca²⁺, K⁺ and Mg²⁺, but completely inhibited by Hg²⁺, Cu²⁺, ionic detergents and phenylmethylsulfonyl fluoride (PMSF) at 0.01 M concentration.     

The relationship between the carboxylesterase and monoacylglycerol lipase activities of chicken liver microsomes

The carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) and monoacylglycerol lipase (glycerol-monoester acylhydrolase, EC 3.1.1.23) activities, measured against ethyl butyrate and emulsified monooleoylglycerol respectively, were determined for chicken liver microsomes and highly purified chicken liver carboxylesterase. The activity ratio (ethyl butyrate activity/monooleoylglycerol activity) was approx. 5 for microsomes and approx. 400 for carboxylesterase. Homogenization of microsomes in 0.1 M Tris-HCl buffer (pH 7.92) released all of the ethyl butyrate activity and about half of the monooleoylglycerol activity into a soluble form. Both activities eluted from a Sephadex G-200 column with the same elution volume as that of pure carboxylesterase. This fraction (fraction B) had an activity ratio of approx. 15, an average pI of 5.01 (cf. 4.75 for carboxylesterase), and ran on polyacrylamide gel electrophoresis at pH 8.6 as a number of closely spaced esterase bands with mobilities considerably less than those of the esterase bands present in the carboxylesterase. Fraction B activities against both substrates were completely inhibited by diethyl p-nitrophenyl phosphate and completely precipitated by antibody to carboxylesterase. The remaining half of the monoacylglycerol lipase activity of microsomes was solubilized by treatment with 1.5% (w/v) Triton X-100. This solubilized monoacylglycerol lipase was completely inhibited by diethyl p-nitrophenyl phosphate, showing it to be a serine-dependent enzyme like the carboxylesterases. However, it had no detectable activity against ethyl butyrate, indicating that it is not closely related to the carboxylesterases.     

Specificity and induction of undecyl acetate esterase from Pseudomonas cepacia grown on 2-tridecanone

Undecyl acetate esterase from Pseudomonas cepacia grown on 2-tridecanone was strongly inhibited by organophosphates and other esterase inhibitors. Also, p-chloromercuribenzoate at 1 x 10(-4) M showed a 70% inhibition of esterase activity. The enzyme hydrolyzed both aliphatic and aromatic acetate esters at substrate concentrations of 0.25 M. Under these conditions the highest reaction rate was toward undecyl acetate. No lipase or proteolytic activity was demonstrated. Undecyl acetate esterase was classified as a carboxylesterase (B-esterase). Cell-free activity studies on the production of undecyl acetate esterase grown on different carbon sources plus zymogram studies demonstrated that the enzyme was inducible when 2-tridecanone, 2-tridecanol, undecyl acetate and, to a lesser extent, 1-undecanol were growth substrates. Induction of undecyl acetate esterase during oxidation of 2-tridecanone supports the view that undecyl acetate is an intermediate in the degradation of the methyl ketone.     

Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp. PDF1

The gene encoding a carboxylesterase from Anoxybacillus sp., PDF1, was cloned and sequenced. The recombinant protein was expressed in Escherichia coli BL21, under the control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme, designated as PDF1Est, was purified by heat shock and ion-exchange column chromatography. The molecular mass of the native protein, as determined by SDS-PAGE, was about 26 kDa. PDF1Est was active under a broad pH range (pH 5.0-10.0) and a broad temperature range (25-90 °C), and it had an optimum pH of 8.0 and an optimum temperature of 60 °C. The enzyme was thermostable carboxylesterase, and did not lose any activity after 30 min of incubation at 60 °C. The enzyme exhibited a high level of activity with p-nitrophenyl butyrate with apparent K(m), V(max), and K(cat) values of 0.348 ± 0.030 mM, 3725.8 U/mg, and 1500 ± 54.50/s, respectively. The effect of some chemicals on the esterase activity indicated that Anoxybacillus sp. PDF1 produce an carboxylesterase having serine residue in active site and -SH groups in specific sites, which are required for its activity.     

EstA from <Emphasis Type="Italic">Arthrobacter nitroguajacolicus</Emphasis> Rü61a,a Thermo- and Solvent-Tolerant Carboxylesterase Related to Class C β-Lactamases

The estA gene encoding a novel cytoplasmic carboxylesterase from Arthrobacter nitroguajacolicus Rü61a was expressed in Escherichia coli. Sequence analysis and secondary structure predictions suggested that EstA belongs to the family VIII esterases, which are related to class C beta-lactamases. The S-x-x-K motif that in beta-lactamases contains the catalytic nucleophile, and a putative active-site tyrosine residue are conserved in EstA. The native molecular mass of hexahistidine-tagged (His6) EstA, purified by metal chelate affinity chromatography, was estimated to be 95 kDa by gel filtration, whereas the His6EstA peptide has a calculated molecular mass of 42.1 kDa. The enzyme catalyzes the hydrolysis of short-chain phenylacyl esters and triglycerides, and shows weak activity toward 2-hydroxy- and 2-nitroacetanilide. Its catalytic activity was inhibited by the serine-specific effector phenylmethylsulfonyl fluoride, and by Cd2+ and Hg2+ ions. Maximum activity of His6EstA was observed at a pH of 9.5 and a temperature of 50 degrees C to 60 degrees C. The enzyme was fairly thermostable. After 19 days at 50 degrees C and after 24 hours at 60 degrees C, its residual relative esterase activity toward phenylacetate was still 53% and 30%, respectively. Exposure of His6EstA to buffer-solvent mixtures showed that the enzyme was inactivated by several high log P (hydrophobic) solvents, whereas it showed remarkable stability and activity in up to 30% (by volume) of polar (low log P) organic solvents such as dimethylsulfoxide, methanol, acetonitrile, acetone, and propanol.     

Purification and characterization of two human liver carboxylesterases

1. Two carboxylesterases (EC 3.1.1.1) purified from human livers were distinguished by pI (isoelectric point), nondenaturing polyacrylamide gel electrophoresis, molecular weight, catalytic activity, N-terminus and immunological cross-reactivity. 2. The low pI carboxylesterase has not been reported previously. 3. Numerous bands seen when each enzyme was focused on analytical IEF gels could not be separated. 4. When sections of the band pattern was refocused, the original complete band pattern was generated. 5. Both the mid and low pI carboxylesterases had catalytic activity for xenobiotics as well as medium and long chain fatty acid esters.     

Comparison of benzil and trifluoromethyl ketone (TFK)-mediated carboxylesterase inhibition using classical and 3D-quantitative structure–activity relationship analysis

Carboxylesterases are enzymes that hydrolyze a broad suite of endogenous and exogenous ester-containing compounds to the corresponding alcohol and carboxylic acid. These enzymes metabolize a number of therapeutics including the anti-tumor agent CPT-11, the anti-viral drug oseltamivir, and the anti-thrombogenic agent clopidogrel as well as many agrochemicals. In addition, carboxylesterases are involved in lipid homeostasis, including cholesterol metabolism and transport with a proposed role in the development of atherosclerosis. Several different scaffolds capable of inhibiting carboxylesterases have been reported, including organophosphates, carbamates, trifluoromethyl ketone-containing structures (TFKs), and aromatic ethane-1,2-diones. Of these varied groups, only the 1,2-diones evidence carboxylesterase isoform-selectivity, which is an important characteristic for therapeutic application and probing biological mechanisms. This study constructed a series of classical and 3D-QSAR models to examine the physiochemical parameters involved in the observed selectivity of three mammalian carboxylesterases: human intestinal carboxylesterase (hiCE), human carboxylesterase 1 (hCE1), and rabbit carboxylesterase (rCE). CoMFA-based models for the benzil-analogs described 88%, 95% and 76% of observed activity for hiCE, hCE1 and rCE, respectively. For TFK-containing compounds, two distinct models were constructed using either the ketone or gem-diol form of the inhibitor. For all three enzymes, the CoMFA ketone models comprised more biological activity than the corresponding gem-diol models; however the differences were small with described activity for all models ranging from 85–98%. A comprehensive model incorporating both benzil and TFK structures described 92%, 85% and 87% of observed activity for hiCE, hCE1 and rCE, respectively. Both classical and 3D-QSAR analysis showed that the observed isoform-selectivity with the benzil-analogs could be described by the volume parameter. This finding was successfully applied to examine substrate selectivity, demonstrating that the relative volumes of the alcohol and acid moieties of ester-containing substrates were predictive for whether hydrolysis was preferred by hiCE or hCE1. Based upon the integrated benzil and TFK model, the next generation inhibitors should combine the A-ring and the 1,2-dione of the benzil inhibitor with the long alkyl chain of the TFK-inhibitor in order to optimize selectivity and potency. These new inhibitors could be useful for elucidating the role of carboxylesterase activity in fatty acid homeostasis and the development of atherosclerosis as well as effecting the controlled activation of carboxylesterase-based prodrugs in situ.