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 by which the enzyme adsorbs to lipid droplets is unclear. Three-dimensional modeling of hTGH predicts that catalytic residues are adjacent to an alpha-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 acid except proline) identified in several other proteins that bind or metabolize neutral lipids. Deletion of this a-helix abolished the lipolytic activity of hTGH. Replacement of F-417 with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L-418 and L-420 with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C-390 is available for covalent acylation. However, neither chemical modification of C-390 nor mutation to alanine affected activity. Our findings indicate that F-417 but not L-418, L-420, or C-390 participates in substrate hydrolysis by hTGH.-Alam, M., D. Gilham, D. E. Vance, and R. Lehner. Mutation of F-417 but not of L-418 or L-420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase.