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)
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. analysis, , -, -, site
- 0. glycosylation, , -, -, site
- 8. glycosylated, , Human TGH, -, -
- 9. N-glycosylation, , -, -, site
- 9. nonglycosylated, , enzyme, -, -
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):