Cloning and sequencing of rat liver carboxylesterase ES-4 (microsomal palmitoyl-CoA hydrolase).
A cDNA which encodes a carboxylesterase of 561 amino acid residues including a cleavable signal peptide is described. The enzyme expressed in COS cells migrates during PAGE (SDS-, and non-denaturing) as a single prominent band in the region of liver ES-4. It ends in the C-terminal cell-retention signal -HNEL, which, in COS cells overexpressing the enzyme, appears to be slightly less efficient than the signals -HTEL and -HVEL of ES-3 and ES-10 respectively. Glycosylation is not essential for intracellular retention, but leads to a higher activity. As do many carboxylesterases, the enzyme expressed in COS cells hydrolyses omicron-nitrophenyl acetate and alpha-naphthyl acetate. It also hydrolyses acetanilide, although less efficiently than ES-3, and, distinctively, palmitoyl-CoA. In addition to the four canonical Cys residues of the carboxylesterases, it contains a fifth, unpaired Cys336, which apparently is not essential for the catalytic properties. Indeed, treatment with iodoacetamide or substitution of Cys336 by Phe does not markedly alter the activity of the enzyme on the various substrates. The predicted structure of ES-4 is highly homologous to that of two other recently cloned esterases which also end in -HNEL [Yan, Yang, Brady and Parkinson (1994) J. Biol. Chem. 269, 29688-29696; Yan, Yang, and Parkinson (1995) Arch. Biochem. Biophys. 317, 222-234]. Together, these isoenzymes probably account for the closely spaced bands observed in the region of ES-4 in non-denaturing PAGE.
Robbi
M
M
Laboratoire de Chimie Physiologique, Université de Louvain, Brussels, Belgium.
Van Schaftingen
E
E
Beaufay
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D007527:Isoenzymes / Q000235:genetics; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010170:Palmitoyl-CoA Hydrolase / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D021382:Protein Sorting Signals; D010170:Palmitoyl-CoA Hydrolase