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Structure−Function Analysis of Human Triacylglycerol Hydrolase by Site-Directed Mutagenesis:  Identification of the Catalytic Triad and a Glycosylation Site

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Departments of Pediatrics and Cell Biology and Department of Biochemistry, CIHR Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
Cite this: Biochemistry 2002, 41, 21, 6679–6687
Publication Date (Web):April 27, 2002
Copyright © 2002 American Chemical Society

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    Triacylglycerol hydrolase is a microsomal enzyme that hydrolyzes stored cytoplasmic triacylglycerol in the liver and participates in the lipolysis/re-esterification cycle during the assembly of very-low-density lipoproteins. The structure−activity relationship of the enzyme was investigated by site-directed mutagenesis and heterologous expression. Expression of human TGH in Escherichia coli yields a protein without enzymatic activity, which suggests that posttranslational processing is necessary for the catalytic activity. Expression in baculovirus-infected Sf-9 cells resulted in correct processing of the N-terminal signal sequence and yielded a catalytically active enzyme. A putative catalytic triad consisting of a nucleophilic serine (S221), glutamic acid (E354), and histidine (H468) was identified. Site-directed mutagenesis of the residues (S221A, E354A, and H468A) yielded a catalytically inactive enzyme. CD spectra of purified mutant proteins were very similar to that of the wild-type enzyme, which suggests that the mutations did not affect folding. Human TGH was glycosylated in the insect cells. Mutagenesis of the putative N-glycosylation site (N79A) yielded an active nonglycosylated enzyme. Deletion of the putative C-terminal endoplasmic reticulum retrieval signal (HIEL) did not result in secretion of the mutant protein. A model of human TGH structure suggested a lipase α/β hydrolase fold with a buried active site and two disulfide bridges (C87−C116 and C274−C285).

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     This research was supported by a grant from the Canadian Institutes of Health Research (UOP-50058) and a contract from GlaxoSmithKline. R.L. is a Heritage Medical Scholar, and D.E.V. is a Heritage Medical Scientist of the Alberta Heritage Foundation for Medical Research.

     Departments of Pediatrics and Cell Biology.


     Department of Biochemistry.


     To whom correspondence should be addressed:  Departments of Pediatrics and Cell Biology, University of Alberta, 328 Heritage Medical Research Centre, Edmonton, Alberta, Canada T6G 2S2. Phone:  (780) 492-2963. Fax:  (780) 492-3383. E-mail:  richard.lehner@

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