Abstract
Human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) constitute a family of mammalian lipases characterized by an acidic pH optimum. HGL and RLL are secreted by the chief cells of the stomach and by the serous von Ebner's glands of the tongue, respectively, and hydrolyze dietary longchain triglycerides in the gastrointestinal tract. HLAL, in contrast, catalyzes the intralysosomal degradation of both triglycerides and cholesteryl esters in virtually all cells except erythrocytes. All three enzymes are proposed to be serine esterases with a catalytic Ser-Asp-His triad similar to other lipases, despite their sensitivity towards sulfhydryl modifying reagents. To investigate the role of conserved serine, aspartic acid, and histidine residues in HLAL and HGL, we constructed 24 mutant lipases with single amino acid substitutions using the site-directed mutagenesis approach. Our combined data strongly support the conclusion that Ser153, Asp324, and His355 are components of the catalytic triad of HLAL and HGL. Structural integrity of the conserved His-Gly dipeptide of lipases also appears to be important for neutral lipid hydrolysis, as replacement of His65 by glutamine abolished HLAL and HGL enzymic activity. Substitution of HLAL residues Asp93, Asp130, and Asp328 with glycine, in contrast, had a more pronounced impact on cholesteryl oleate hydrolysis than on triglyceride hydrolysis. These results provide new insights into the structural basis of HLAL and HGL function.
Highlights
Human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) constitute a family of mammalian lipases characterized by an acidic pH optimum
Asp,:, Asp, and Aspspxwith glycine, in contrast, had a more pronounced impact on cholesteryl oleate hydrolysis than on triglyceride hydrolysis. These results provide new insights into the structural basis of human lysosomal acid lipase (HLAL), and HGL function.-Lohse, P., S
In most lipases, the active site serine residue is surrounded by two invariant glycines in order to maintain the tight bend between the P-strand and the &helix [8, 9], it appears highly unlikely that one of these amino acids is the nucleophile of the catalytic triad
Summary
Human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) constitute a family of mammalian lipases characterized by an acidic pH optimum. Asp,:,, Asp,,,,, and Aspspxwith glycine, in contrast, had a more pronounced impact on cholesteryl oleate hydrolysis than on triglyceride hydrolysis These results provide new insights into the structural basis of HLAL, and HGL function.-Lohse, P., S. Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: identification of the catalytically active serine, aspartic acid, and histidine residues. With the exception of a lipase/acyltransferase from Aeromonas hydrqbhilu [6,7],the active site serine is always part of the conserved pentapeptide motif -Gly-XaaSer-Xaa-Gly- This lipase consensus sequence forms a loop structure with the serine residue connecting a pstrand to an a-helix [8].This D-ESer-a conformation, known as the “nucleophilic elbow” [9],facilitates interaction of the nucleophile with the histidine and the ester carbon in a precisely oriented complex. Lipases and cholesteryl esterases catalyze the hydrolysis of bonds in neutral lipids
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