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PubMed1983

Human liver carboxylesterase. Properties and comparison with human serum carboxylesterase.

Carboxylesterase was obtained from human liver in an electrophoretically homogeneous form. The monomeric molecular weight of the enzyme was 60,000 and the enzyme associated to form trimers. Purified human liver carboxylesterase was compared with human serum carboxylesterase, purified earlier. Serum carboxylesterase hydrolyzed a typical cholinesterase substrate and aryl acylamide, whereas liver carboxylesterase did not hydrolyze these compounds. Both carboxylesterases catalyzed the hydrolysis of short-chain triacylglycerols, such as tributyrin, and medium-chain monoacylglycerols, such as monocaprin, but not the hydrolysis of long-chain triacylglycerols. Serum carboxylesterase activity was inhibited by p-trimethylammoniumanilinium dichloride and neostigmine, whereas liver carboxylesterase activity was not affected by these compounds. Liver and serum carboxylesterase activities were both strongly inhibited by phenylmethylsulfonyl fluoride.

Carboxylic Ester Hydrolases↗

PubMed2004

Hydrolysis of irinotecan and its oxidative metabolites, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin and 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin, by human carboxylesterases CES1A1, CES2, and a newly expressed carboxylesterase isoenzyme, CES3.

Carboxylesterases metabolize ester, thioester, carbamate, and amide compounds to more soluble acid, alcohol, and amine products. They belong to a multigene family with about 50% sequence identity between classes. CES1A1 and CES2 are the most studied human isoenzymes from class 1 and 2, respectively. In this study, we report the cloning and expression of a new human isoenzyme, CES3, that belongs to class 3. The purified recombinant CES3 protein has carboxylesterase activity. Carboxylesterases metabolize the carbamate prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11; irinotecan) to its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I inhibitor. CYP3A4 oxidizes CPT-11 to two major oxidative metabolites, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin (APC) and 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin (NPC). In this study, we investigate whether these oxidative metabolites, NPC and APC, can be metabolized to SN-38 by purified human carboxylesterases, CES1A1, CES2, and CES3. We find that CPT-11, APC, and NPC can all be metabolized by carboxylesterases to SN-38. CES2 has the highest catalytic activity of 0.012 min(-1) microM(-1) among the three carboxylesterases studied for hydrolysis of CPT-11. NPC was an equally good substrate of CES2 in comparison to CPT-11, with a catalytic efficiency of 0.005 min(-1) microM(-1). APC was a very poor substrate for all three isoenzymes, exhibiting a catalytic activity of 0.015 x 10(-3) min(-1) microM(-1) for CES2. Catalytic efficiency of CES3 for CPT-11 hydrolysis was 20- to 2000-fold less than that of CES1A1 and CES2. The relative activity of the three isoenzymes was CES2 > CES1A1 >> CES3, for all three substrates.

Amino Acid Sequence↗

PubMed2004

Characterization of inhibitors of specific carboxylesterases: development of carboxylesterase inhibitors for translational application.

Carboxylesterases, expressed at high levels in human liver and intestine, are thought to detoxify xenobiotics. The anticancer prodrug 7-ethyl-10-[4-1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) is also metabolized by carboxylesterases to produce the active drug 7-ethyl-10-hydroxycamptothecin. Activation of CPT-11 by human intestinal carboxylesterase (hiCE) in the human intestine may contribute to delayed onset diarrhea, a dose-limiting side effect of this drug. The goal of this study was to develop small molecule inhibitors selective for hiCE to circumvent or treat the toxic side effects of CPT-11. A secondary goal was to develop molecules that specifically inhibit activation of CPT-11 by a rabbit liver carboxylesterase (rCE). rCE is the most efficient CPT-11-activating enzyme thus far identified, and this enzyme is being developed for viral-directed enzyme prodrug therapy applications. Based on in vitro assays with partially purified hiCE and rCE proteins and on growth inhibition assays using U373MG human glioma cells transfected to express hiCE or rCE (U373pIREShiCE or U373pIRESrCE), we identified specific inhibitors of each enzyme. Lead compounds are derivatives of nitrophenol having 4-(furan-2-carbonyl)-piperazine-1-carboxylic acid or 4-[(4-chlorophenyl)-phenylmethyl]-piperazine-1-carboxylic acid substitutions in the p position. Kinetic analysis of each compound for hiCE compared with rCE showed that the Ki values of the most selective of these inhibitors differed by 6- to 10-fold. In growth inhibition assays, nontoxic, low micromolar concentrations of these inhibitors increased the EC50 of CPT-11 for U373pIREShiCE or U373pIRESrCE cells by 13- to >1,500-fold. The four compounds characterized in this study will serve as lead compounds for a series of inhibitors to be constructed using a combinatorial approach.

Animals↗

PubMed1975

Carboxylesterases (EC 3.1.1). The molecular sizes of chicken and pig liver carboxylesterases.

The molecular size of pig liver carboxylesterase has been investigated under a variety of conditions of pH and ionic strength. From equilibrium and velocity sedimentation at pH 4.0 and pH 7.5, and from chromatography on Sephadex G-200,we conclude that the monomeric molecular weight is similar to 65,000 daltons and that the enzyme associates to form trimers. Association equilibrium constants for the monomer-trimer system were estimated to be 0.02 1-2 g-2 at pH 4 (concentration-dependent molecular weight data) and 2 times 10-5 1-2g-2 at pH 7.5 (frontal gel chromatographic results). These studies were aided by comparisons of the properties of the pig liver enzyme with those of chicken liver carboxylesterase, which is shown to exhibit the velocity and equilibrium sedimentation characteristics of a homogeneous protein with molecular weight similar to 65,000. Studies of pig and chicken liver carboxylesterases in 6 M guanidinium chloride, 0.1 M in beta-mercaptoethanol, support the proposition that the monomeric species of these enzymes have molecular weights of similar to 65,000. On polyacrylamide gel electrophoresis in SDS, there is no evidence for a major species of molecular weight less than similar to 65,000 for the pig enzyme, but ca. 50 percent of the chicken esterase is dissociated into two species of molecular weight similar to 30,000.

Animals↗

PubMed1993

Isolation and properties of carboxylesterases of the termite gut-associated fungus, Xylaria nigripes. K., and their identity from the host termite, Odentotermes horni. W., mid-gut carboxylesterases.

1. The termite, Odentotermes horni. W., houses three fungal species, viz. Xylaria nigripes, Termitomyces microcorpus, and Trichoderma (species not identified), in its gut. X. nigripes was found to possess higher esterase activity levels than the other two. 2. Four esterase enzymes, viz. FE-I, -II, -III and -IV, with pI values 5.1, 5.25, 5.4 and 5.6, respectively, were identified, isolated and purified to apparent homogeneity from the fungus X. nigripes, their biochemical and enzymological properties were determined, and compared with those of the previously characterized host termite mid-gut enzymes, TE-I and -II. 3. The M(r) of FE-I and -II was 85.1 kDa and those of FE-III and -IV was 87.5 kDa. However, TE-I and -II were relatively smaller (M(r) approximately 78.5 kDa). Each of the fungal enzymes, viz. FE-I to -IV, was a homodimer with subunits associated non-covalently. The subunit M(r) were 42.6 kDa for FE-I and -II, and 43.7 kDa for FE-III and -IV. On the other hand, the termite mid-gut enzymes, TE-I and -II, were also homodimeric, but the subunits were associated covalently (subunit M(r) = 40 kDa). Immunologically the fungal esterase enzymes, viz. FE-I to -IV, were different from those of the host termite mid-gut esterases, viz. TE-I and -II. 4. The substrate specificity and inhibitor sensitivity studies classify these enzymes, i.e. FE-I to -IV, as carboxylesterases (EC 3.1.1.1). Steady-state product inhibition kinetics suggested; an ordered release of products, i.e. alcohol followed by acid, and a Uni-Bi kinetic reaction mechanism. 5. The two preliminary studies, i.e. the confinement of most esterase activity to the gut-tissue free from microorganisms and starvation of termites not leading to complete loss of esterase activity in the gut of the termites, suggested that there may not be any symbiotic relationship between termite, O. horni, and its gut associated microorganisms with regard to ester metabolism. Though the enzymes from the two sources were carboxylesterases, several of their properties were different and hence, they are different entities.

Amino Acids↗

PubMed2006

Anti-influenza prodrug oseltamivir is activated by carboxylesterase human carboxylesterase 1, and the activation is inhibited by antiplatelet agent clopidogrel.

Oseltamivir is the main medicine recommended by the World Health Organization in anticipation of next influenza pandemic. This anti-influenza viral agent is an ester prodrug, and the antiviral activity is achieved by its hydrolytic metabolite: oseltamivir carboxylate. In this study, we report that the hydrolytic activation is catalyzed by carboxylesterase human carboxylesterase (HCE) 1. Liver microsomes rapidly hydrolyzed oseltamivir, but no hydrolysis was detected with intestinal microsomes or plasma. The overall rate of the hydrolysis varied among individual liver samples and was correlated well with the level of HCE1. Recombinant HCE1 but not HCE2 hydrolyzed this prodrug and produced similar kinetic parameters as the liver microsomes. Several HCE1 natural variants differed from the wild-type enzyme on the hydrolysis of oseltamivir. In the presence of antiplatelet agent clopidogrel, the hydrolysis of oseltamivir was inhibited by as much as 90% when the equal concentration was assayed. Given the fact that hydrolysis of oseltamivir is required for its therapeutic activity, concurrent use of both drugs would inhibit the activation of oseltamivir, thus making this antiviral agent therapeutically inactive. This is epidemiologically of significance because people who receive oseltamivir and clopidogrel simultaneously may maintain susceptibility to influenza infection or a source of spreading influenza virus if already infected.

Antiviral Agents↗

PubMed1975

Carboxylesterases (EC 3.1.1). A comparison of some kinetic properties of horse, sheep, chicken, pig, and ox liver carboxylesterases.

A comparative study of the kinetic be,avior of horse, sheep, chicken, pig, and ox liver carboxylesterases is reported. The enzymes exhibit similar specificites towards a series of phenyl esters in which the acyl group is varied, and towards a series of butyrate esters in which the alcohol group is varied. Non-Michaelis-Menten kinetics are exhibited by the horse enzyme in the hydrolysis of methyl and ethyl butyrates, and by the pig enzyme with ethyl butyrate. Each enzyme exhibits inhibition by one or more substrates. A simple scheme which accounts for both activation and inhibition is discussed. pH-k(cat) profiles for the horse and chicken liver carboxylesterase-catalyzed hydrolyses of phenyl butyrate demonstrate dependencies on pK(a)S of 4.75 and 5.0, respectively.

Animals↗

PubMed1980

Differential expression of allelic carboxylesterase-2 genes in oocytes of loach (Misgurnus fossilis L.) and heterogeneity of loach oocytes and eggs for the expression of allelic carboxylesterase-2 genes.

Tissue and cell differences have been found in the expression of allelic carboxylesterase-2 (E-2) (carboxylic ester hydrolase, E.C. 3.1.1.1) genes of the loach. The relative activity of two allozymes in the brain and muscles of heterozygotes is similar, whereas in oocytes and eggs of the same fish the activity of one of the allozymes is considerably greater than that of the other. Oocytes and eggs of some heterozygotes were shown to be heterogeneous for the expression of E-2 alleles. The phenomenon implies that roe produced by a heterozygous female can contain two or more egg types: in some the alleles are equally expressed, while in others one of the alleles is predominantly expressed.

Alleles↗

PubMed2000

Characterization of CPT-11 converting carboxylesterase activity in colon tumor and normal tissues: comparison with p-nitro-phenylacetate converting carboxylesterase activity.

Irinotecan (CPT-11) is a topoisomerase I inhibitor commonly used in the treatment of colorectal tumors. It is a prodrug, converted to an active metabolite, SN-38, by carboxylesterases (CEs). CEs are ubiquitary enzymes that react with numerous substrates. A specific CPT-11 converting enzyme was isolated from rat serum, with different kinetic properties than other CEs. We determined kinetic properties of specific CPT-11 CE activity (CPT-CE) in human normal liver and colon tumors. Km were very similar (3.4 microM in liver and 3.8 microM in colon tumors), but Vmax was higher in liver (2.7 pmol/min/mg protein) than in colon tumor (1.7 pmol/min/mg protein). CPT-CE and total CE (using p-nitro-phenylacetate as substrate) were weakly correlated in colon tumors. The large interpatient variability observed in liver CPT-CE activity could play a potential role in the pharmacokinetic variability observed with irinotecan.

Animals↗

PubMed1975

Carboxylesterases (EC 3.1.1). Purification and titration of chicken, sheep, and horse liver carboxylesterases.

Chicken, sheep, and horse liver carboxylesterases have been purified by procedures involving ammonium sulfate fractionation, ion-exchange chromatography and gel filtration on Sephadex. The actual yields of the procedures described were as follows: chicken, 1 g from 2 kg of liver powder (chloroform-acetone); sheep, 200 mg from 400 g of powder (chloroform-acetone); horse, 230 mg from 800 g of powder (acetone). The purified enzymes are free of non-carboxyl-esterase protein as shown by gel electrophoresis, although they do contain electrophoretic variants. The equivalent weight of the chicken enzyme is 67,000 based on titration with p-nitrophenyl diethyl phosphate or bis(p-nitrophenyl) phosphate, whereas those of the sheep and horse enzymes are similar to 69,500 and similar to 70,000, respectively, based on titration with p-nitrophenyl dimethylcarbamate.

Ammonium Sulfate↗

PubMed1975

Carboxylesterases (EC 3.1.1). Amino acid composition of liver carboxylesterases.

The amino acid compositions of the carboxylesterases from chicken, ,orse, ox, sheep, and pig livers are reported and compared. As would be expected for this homologous series, the compositions show a general similarity. However, there are some significant differences, but the degree to which particular pairs of enzymes differ is consistent with the evolutionary history of the species from which they were isolated.

Amino Acids↗

PubMed1975