title: Studies on the reaction mechanism of a microbial lipase/acyltransferase using chemical modification and site-directed mutagenesis.
abstract: Aeromonas hydrophila releases a protein which is a member of the lipase superfamily, similar in reaction mechanism to the important mammalian plasma enzyme lecithin-cholesterol acyltransferase. We have used chemical modification and site-directed mutagenesis of the protein to identify amino acids which may be involved in catalysis. The enzyme was unaffected by phenylmethylsulfonyl fluoride, but it was almost completely inhibited by another serine-reactive compound, diethyl p-nitrophenyl phosphate. A serine selectively modified by this reagent was identified by sequencing the amino-terminal region of the protein. It was located at position 16, in the short consensus sequence shared by the enzyme with other lipases. When this serine was changed to asparagine the product was an enzymatically inert protein which nevertheless retained the surface activity of the wild-type enzyme, suggesting its ability to bind to substrate was not changed. Diethylpyrocarbonate treatment drastically reduced the rate of acyl transfer by the native enzyme, but this did not appear to be due to modification of an essential histidine, since inhibition was not reversed by addition of hydroxylamine. We have shown that only two of the histidines in the enzyme can be involved in catalysis (Hilton, S., McCubbin, W. D., Kay, C.M., and Buckley, J. T. (1990) Biochemistry, 29, 9072-9078). Replacing both of these with asparagines had little or no effect on enzyme activity. These results indicate that, in apparent contrast to other lipases, histidine does not participate in the reaction catalyzed by the microbial enzyme. Since catalysis was not inhibited by sulfhydryl reagents, we conclude that a free cysteine is also not required for activity. This may distinguish the microbial enzyme from the mammalian acyltransferase.
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Hilton
authors.forename: J T
authors.initials: JT
authors.lastname: Buckley
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000333:Aeromonas / Q000201:enzymology*; D000596:Amino Acids / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008049:Lipase / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000378:metabolism; D010957:Plasmids
chemical_list: D000596:Amino Acids; D010713:Phosphatidylcholines; D000217:Acyltransferases; C020597:glycerophospholipid-cholesterol acyltransferase; D008049:Lipase