Mutation of Residues 423 (Met/Ile), 444 (Thr/Met), and 506 (Asn/Ser) Confer Cholesteryl Esterase Activity on Rat Lung Carboxylesterase

Ile(423) alone or in combination with other mutations increased activity with p-nitrophenylcaprylate (PNPC) relative to more hydrophilic p-nitrophenylacetate (PNPA), typical of hncCEH. The substitution Thr(444) --> Met(444) was necessary but not sufficient for expression of cholesteryl esterase activity in COS-7 cells. The substitution Asn(506) --> Ser(506), creating a potential phosphorylation site, uniformly increased activity with both PNPA and PNPC, was necessary but not sufficient for expression of cholesteryl esterase activity and conferred susceptibility to activation by cAMP-dependent protein kinase, a property of hncCEH. The 3 mutations in combination were necessary and sufficient for expression of cholesteryl esterase activity by the mutated LCE. The substitution Gln(186) --> Arg(186) selectively reduced esterase activity with PNPA and PNPC but was not required for cholesteryl esterase activity. Homology modeling from x-ray structures of acetylcholinesterases is used to propose three-dimensional models for hncCEH and LCE that provide insight into the effects of these mutations on substrate specificity. -->
ISSN: 0021-9258
DOI: 10.1074/jbc.m105644200
Přístupová URL adresa: https://explore.openaire.eu/search/publication?articleId=doi_________::d9feebb8d85a9eb83b31836393e94a2f
https://doi.org/10.1074/jbc.m105644200
Rights: OPEN
Přírůstkové číslo: edsair.doi...........d9feebb8d85a9eb83b31836393e94a2f
Autor: Timothy J. Wallace, William M. Grogan, Timothy B. Langston, Mervat R. Gergis, Ehab M. Kodsi
Rok vydání: 2001
Předmět:
Zdroj: Journal of Biological Chemistry. 276:33165-33174
ISSN: 0021-9258
DOI: 10.1074/jbc.m105644200
Popis: Site-directed mutagenesis is used to identify amino acid residues that dictate reported differences in substrate specificity between rat hepatic neutral cytosolic cholesteryl ester hydrolase (hncCEH) and rat lung carboxylesterase (LCE), proteins differing by only 4 residues in their primary sequences. Beginning with LCE, the substitution Met(423) --> Ile(423) alone or in combination with other mutations increased activity with p-nitrophenylcaprylate (PNPC) relative to more hydrophilic p-nitrophenylacetate (PNPA), typical of hncCEH. The substitution Thr(444) --> Met(444) was necessary but not sufficient for expression of cholesteryl esterase activity in COS-7 cells. The substitution Asn(506) --> Ser(506), creating a potential phosphorylation site, uniformly increased activity with both PNPA and PNPC, was necessary but not sufficient for expression of cholesteryl esterase activity and conferred susceptibility to activation by cAMP-dependent protein kinase, a property of hncCEH. The 3 mutations in combination were necessary and sufficient for expression of cholesteryl esterase activity by the mutated LCE. The substitution Gln(186) --> Arg(186) selectively reduced esterase activity with PNPA and PNPC but was not required for cholesteryl esterase activity. Homology modeling from x-ray structures of acetylcholinesterases is used to propose three-dimensional models for hncCEH and LCE that provide insight into the effects of these mutations on substrate specificity.
Databáze: OpenAIRE
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