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

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• 作者：Mustafa Alam ; Dennis E. Vance ; and Richard Lehner
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：May 28, 2002
• 年：2002
• 卷：41
• 期：21
• 页码：6679 - 6687
• 全文大小：489K
• 年卷期：v.41,no.21(May 28, 2002)
• ISSN：1520-4995

文摘

Triacylglycerol hydrolase is a microsomal enzyme that hydrolyzes stored cytoplasmictriacylglycerol in the liver and participates in the lipolysis/re-esterification cycle during the assembly ofvery-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 yieldsa protein without enzymatic activity, which suggests that posttranslational processing is necessary for thecatalytic activity. Expression in baculovirus-infected Sf-9 cells resulted in correct processing of theN-terminal signal sequence and yielded a catalytically active enzyme. A putative catalytic triad consistingof a nucleophilic serine (S221), glutamic acid (E354), and histidine (H468) was identified. Site-directedmutagenesis of the residues (S221A, E354A, and H468A) yielded a catalytically inactive enzyme. CDspectra of purified mutant proteins were very similar to that of the wild-type enzyme, which suggests thatthe mutations did not affect folding. Human TGH was glycosylated in the insect cells. Mutagenesis of theputative N-glycosylation site (N79A) yielded an active nonglycosylated enzyme. Deletion of the putativeC-terminal endoplasmic reticulum retrieval signal (HIEL) did not result in secretion of the mutant protein.A model of human TGH structure suggested a lipase / hydrolase fold with a buried active site and twodisulfide bridges (C87-C116 and C274-C285).