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Journal of Lipid Research, Vol. 47, 375-383, February 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

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

Mustafa Alam^*, Dean Gilham, Dennis E. Vance and Richard Lehner^1,*,

^* Department of Pediatrics, Canadian Institutes of Health Research Group on the Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
Department of Cell Biology, Canadian Institutes of Health Research Group on the Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
Department of Biochemistry, Canadian Institutes of Health Research Group on the Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta, Canada T6G 2S2

Published, JLR Papers in Press, November 10, 2005.

¹ To whom correspondence should be addressed. e-mail: richard.lehner{at}ualberta.ca

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 -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 -helix abolished the lipolytic activity of hTGH. Replacement of F₄₁₇ with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L₄₁₈ and L₄₂₀ with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C₃₉₀ is available for covalent acylation. However, neither chemical modification of C₃₉₀ nor mutation to alanine affected activity. Our findings indicate that F₄₁₇ but not L₄₁₈, L₄₂₀, or C₃₉₀ participates in substrate hydrolysis by hTGH.

Supplementary key words lipolysis • mutagenesis • carboxylesterase • lipase

Abbreviations: hCETP, human plasma cholesteryl ester transfer protein; hTGH, human triacylglycerol hydrolase; 4MUH, 4-methylumbelliferyl heptanoate; Ni-NTA, nickel-nitrilotriacetic acid; NLBD, neutral lipid binding domain; PNP, p-nitrophenyl; TG, triacylglycerol; TGH, triacylglycerol hydrolase

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