Purification and characterization of guinea-pig liver microsomal deacetylase involved in the deacetylation of the O-glucoside of N-hydroxyacetanilide

Suzuki-Kurasaki, Mika; Yoshioka, Takayuki; Uematsu, Takayoshi

doi:10.1042/bj3250155

Cited by 10 publications

(4 citation statements)

References 47 publications

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“…For example, Suzuki-Kurasaki reported that the guinea pig liver microsomal deacylase is a hexameric protein, consisting of two trimers and six substrate-binding sites. This enzyme also exhibited allosteric enzyme kinetics with different substrates (31).…”

Section: Discussionmentioning

confidence: 85%

Molecular Modeling of CPT-11 Metabolism by Carboxylesterases (CEs): Use of pnb CE as a Model

et al. 2004

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CPT-11 is a prodrug that is converted in vivo to the topoisomerase I poison SN-38 by carboxylesterases (CEs). Among the CEs studied thus far, a rabbit liver CE (rCE) converts CPT-11 to SN-38 most efficiently. Despite extensive sequence homology, however, the human homologues of this protein, hCE1 and hiCE, metabolize CPT-11 with significantly lower efficiencies. To understand these differences in drug metabolism, we wanted to generate mutations at individual amino acid residues to assess the effects of these mutations on CPT-11 conversion. We identified a Bacillus subtilis protein (pnb CE) that could be used as a model for the mammalian CEs. We demonstrated that pnb CE, when expressed in Escherichia coli, metabolizes both the small esterase substrate o-NPA and the bulky prodrug CPT-11. Furthermore, we found that the pnb CE and rCE crystal structures show an only 2.4 A rmsd variation over 400 residues of the alpha-carbon trace. Using the pnb CE model, we demonstrated that the "side-door" residues, S218 and L362, and the corresponding residues in rCE, L252 and L424, were important in CPT-11 metabolism. Furthermore, we found that at position 218 or 252 the size of the residue, and at position 362 or 424 the hydrophobicity and charge of the residue, were the predominant factors in influencing drug activation. The most significant change in CPT-11 metabolism was observed with the L424R variant rCE that converted 10-fold less CPT-11 than the wild-type protein. As a result, COS-7 cells expressing this mutant were 3-fold less sensitive to CPT-11 than COS-7 cells expressing the wild-type protein.

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Section: Discussionmentioning

confidence: 85%

Molecular Modeling of CPT-11 Metabolism by Carboxylesterases (CEs): Use of pnb CE as a Model

et al. 2004

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“…Fitting the data to an allosteric model by non-linear regression gave values for the Hill coefficient ( h ) for both enzymes >1.00 ( Table 2 ). It is interesting to note that native pig and human liver microsomal carboxylesterases have previously been reported to exhibit positive cooperativity [ 44 , 45 ]. This phenomenon is typical of enzymes comprising more than one identical subunit and can be interpreted by reference to one of two models namely the Monod-Wyman-Changeux (MWC) concerted model [ 46 ] and the Koshland-Nemethy-Filmer (KNF) sequential model [ 47 ].…”

Section: Resultsmentioning

confidence: 99%

“…Thus transitions from low to high substrate binding affinity may be modulated by substrate binding to a non-catalytic site on the enzyme. Most interestingly, analysis of the kinetics of natural pig microsomal carboxylesterase 1 according to a concerted model of cooperativity suggested that each subunit of the trimeric enzyme had two substrate binding sites [ 45 ]. Overall the enzyme assay results confirm that N-glycosylation does not affect binding of the 4-NPA substrate and are consistent with recent results for the hCES1 expressed in E .…”

Section: Resultsmentioning

confidence: 99%

Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1

et al. 2015

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Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme.

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“…The binding of ligands to a surface Z-site on hCE1 shifts the trimer-hexamer equilibrium to trimer. Thus this surface position likely functions as an allosteric binding site, as has been proposed for a related mammalian carboxylesterase [25].…”

Section: Heroin and Cocaine Metabolism By Hce1mentioning

confidence: 90%

Human carboxylesterase 1: from drug metabolism to drug discovery

Redinbo

Bencharit

Potter

2003

Biochemical Society Transactions

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Human carboxylesterase 1 (hCE1) is a serine esterase involved in both drug metabolism and activation, as well as other biological processes. hCE1 catalyses the hydrolysis of heroin and cocaine, and the transesterification of cocaine in the presence of ethanol to the toxic metabolite cocaethylene. We have determined the crystal structures of hCE1 in complex with either the cocaine analogue homatropine or the heroin analogue naloxone. These are the first structures of a human carboxylesterase, and they provide details about narcotic metabolism in humans. hCE1's active site contains rigid and flexible pockets, explaining the enzyme's ability to act both specifically and promiscuously. hCE1 has also been reported to contain cholesteryl ester hydrolase, fatty acyl-CoA hydrolase and acyl-CoA:cholesterol acyltransferase activities, and thus appears to be involved in cholesterol metabolism. Since the enzyme may be useful as a treatment for cocaine overdose, and may afford protection against chemical weapons like Sarin, Soman and VX gas, hCE1 could serve as both a drug and a drug target. Selective hCE1 inhibitors targeted to several sites on the enzyme may also pave the way for novel clinical tools to manage cholesterol homoeostasis in humans.

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Purification and characterization of guinea-pig liver microsomal deacetylase involved in the deacetylation of the O-glucoside of N-hydroxyacetanilide

Cited by 10 publications

References 47 publications

Molecular Modeling of CPT-11 Metabolism by Carboxylesterases (CEs): Use of pnb CE as a Model

Molecular Modeling of CPT-11 Metabolism by Carboxylesterases (CEs): Use of pnb CE as a Model

Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1

Human carboxylesterase 1: from drug metabolism to drug discovery

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