Mutation of F417 but not of L418 or L420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase

Human triacylglycerol hydrolase (hTGH) has been shown to play a role in hepatic lipid metabolism. Triacylglycerol hydrolase (TGH) hydrolyzes insoluble carboxylic esters at lipid/water interfaces, although the mechanism bywhich the enzyme adsorbs to lipid droplets is unclear. Three-dimensional modeling of hTGH predicts that catalytic residues are adjacent to an α-helix that may mediate TGH/lipid interaction. The helix contains a putative neutral lipid binding domain consisting of the octapeptide FLDLIADV (amino acid residues 417-424) with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino add except proline) identified in several other proteins that bind or metabolize neutral lipids. Deletion of this α-helix abolished the lipolytic activity of hTGH. Replacement of F 4 1 7 with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L 4 1 8 and L 4 2 0 with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C 3 9 0 is available for covalent acylation. However, neither chemical modification of C 3 9 0 nor mutation to alanine affected activity. Our findings indicate that F 4 1 7 but not L 4 1 8 , L 4 2 0 , or C 3 9 0 Participates in substrate hydrolysis by hTGH.