Identification of the active site serine in pancreatic cholesterol esterase by chemical modification and site-specific mutagenesis.
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.
DiPersio
L P
LP
Department of Pathology, University of Cincinnati College of Medicine, Ohio 45267-0529.
Fontaine
R N
RN
Hui
D Y
DY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010179:Pancreas / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D002787:Sterol Esterase / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D002787:Sterol Esterase