title: Structure-function analysis of human triacylglycerol hydrolase by site-directed mutagenesis: identification of the catalytic triad and a glycosylation site.
abstract: 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 alpha/beta hydrolase fold with a buried active site and two disulfide bridges (C87-C116 and C274-C285).
authors.affiliation: Department of Pediatrics, CIHR Group on Molecular and Cell Biology of Lipids, University of Alberta, 328 Heritage Medical Centre, Edmonton, Alberta, Canada T6G 2S2.
authors.forename: Mustafa
authors.initials: M
authors.lastname: Alam
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Vance
authors.forename: Richard
authors.initials: R
authors.lastname: Lehner
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007313:Insecta / Q000235:genetics / Q000378:metabolism; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D018698:Glutamic Acid; D012694:Serine; D006639:Histidine; D008049:Lipase