DiPersio_1990_J.Biol.Chem_265_16801

Reference

Title : Identification of the active site serine in pancreatic cholesterol esterase by chemical modification and site-specific mutagenesis - DiPersio_1990_J.Biol.Chem_265_16801
Author(s) : DiPersio LP , Fontaine RN , Hui DY
Ref : Journal of Biological Chemistry , 265 :16801 , 1990
Abstract :

Chemical modification and site-specific mutagenesis approaches were used in this study to identify the active site serine residue of pancreatic cholesterol esterase. In the first approach, purified porcine pancreatic cholesterol esterase was covalently modified by incubation with [3H]diisopropylfluorophosphate (DFP). The radiolabeled cholesterol esterase was digested with CNBr, and the peptides were separated by high performance liquid chromatography. A single 3H-containing peptide was obtained for sequence determination. The results revealed the binding of DFP to a serine residue within the serine esterase homologous domain of the protein. Furthermore, the DFP-labeled serine was shown to correspond to serine residue 194 of rat cholesterol esterase (Kissel, J. A., Fontaine, R. N., Turck, C. W., Brockman, H. L., and Hui, D. Y. (1989) Biochim. Biophys. Acta 1006, 227-236). The codon for serine 194 in rat cholesterol esterase cDNA was then mutagenized to ACT or GCT to yield mutagenized cholesterol esterase with either threonine or alanine, instead of serine, at position 194. Expression of the mutagenized cDNA in COS-1 cells demonstrated that substitution of serine 194 with threonine or alanine abolished enzyme activity in hydrolyzing the water-soluble substrate, p-nitrophenyl butyrate, and the lipid substrates cholesteryl [14C]oleate and [14C] lysophosphatidylcholine. These studies definitively identified serine 194 in the catalytic site of pancreatic cholesterol esterase.

PubMedSearch : DiPersio_1990_J.Biol.Chem_265_16801
PubMedID: 2211595
Gene_locus related to this paper: ratno-balip

Related information

Gene_locus ratno-balip

Citations formats

DiPersio LP, Fontaine RN, Hui DY (1990)
Identification of the active site serine in pancreatic cholesterol esterase by chemical modification and site-specific mutagenesis
Journal of Biological Chemistry 265 :16801

DiPersio LP, Fontaine RN, Hui DY (1990)
Journal of Biological Chemistry 265 :16801