Site-specific mutagenesis of an essential histidine residue in pancreatic cholesterol esterase.
The histidine residue essential for the catalytic activity of pancreatic cholesterol esterase (carboxylester lipase) has been identified in this study using sequence comparison and site-specific mutagenesis techniques. In the first approach, comparison of the primary structure of rat pancreatic cholesterol esterase with that of acetylcholinesterase and cholinesterase revealed two conserved histidine residues located at positions 420 and 435. The sequence in the region around histidine 420 is quite different between the three enzymes. However, histidine 435 is located in a 22-amino acid domain that is 47% homologous with other serine esterases. Based on this sequence homology, it was hypothesized that histidine 435 is the histidine residue essential for catalytic activity of cholesterol esterase. The role of His435 in the catalytic activity of pancreatic cholesterol esterase was then studied by the site-specific mutagenesis technique. Substitution of the histidine in position 435 with glutamine, arginine, alanine, serine, or aspartic acid abolished the ability of cholesterol esterase to hydrolyze p-nitrophenyl butyrate and cholesterol [14C]oleate. In contrast, mutagenesis of the histidine residue at position 420 to glutamine had no effect on cholesterol esterase enzyme activity. The results of this study strongly suggested that histidine 435 may be a component of the catalytic triad of pancreatic cholesterol esterase.
DiPersio
L P
LP
Department of Pathology and Laboratory Medicine, 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; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D005810:Multigene Family; D009841:Oligonucleotides / Q000737:chemistry; D010179:Pancreas / Q000201:enzymology*; D051381:Rats; D002787:Sterol Esterase / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection
D009841:Oligonucleotides; D006639:Histidine; D002787:Sterol Esterase