Identification of the active site serine of hormone-sensitive lipase by site-directed mutagenesis.
The consensus pentapeptide GXSXG is found in virtually all lipases/esterases and generally contains the active site serine. The primary sequence of hormone-sensitive lipase contains a single copy of this pentapeptide, surrounding Ser-423. We have analyzed the catalytic role of Ser-423 by site-directed mutagenesis and expression of the mutant hormone-sensitive lipase in COS cells. Substitution of Ser-423 by several different amino acids resulted in the complete abolition of both lipase and esterase activity, whereas mutation of other conserved serine residues had no effect on the catalytic activity. These results strongly suggest that Ser-423 is the active site serine of hormone-sensitive lipase.
Holm
C
C
Department of Medical and Physiological Chemistry, Lund University, Sweden.
Davis
R C
RC
Osterlund
T
T
Schotz
M C
MC
Fredrikson
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D051381:Rats; D012694:Serine / Q000235:genetics / Q000378:metabolism; D002787:Sterol Esterase / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection
D010446:Peptide Fragments; D012694:Serine; D002787:Sterol Esterase