Identification of a domain of lecithin-cholesterol acyltransferase that is involved in interfacial recognition.
Lecithin-cholesterol acyltransferase (LCAT) is an interfacial enzyme that acts on lipid substrates on the surface of high density lipoproteins (HDL). Based on observations with other interfacial lipases, we propose that LCAT contains a surface region of 25 amino acids linked by a disulfide bond (C50-C74) that is involved in the binding of LCAT to lipoproteins. Using LCAT cDNA, we have deleted most of this region (delta 53-71) and expressed the mutant enzyme (LCAT delta 53-71) in COS-1 cells. The deletion mutant is expressed and secreted at levels similar to wildtype LCAT, suggesting that the deleted region is located on the surface of the enzyme and is not required for folding. The enzymatic activity of the mutant was tested using two interfacial substrates, reconstituted HDL (rHDL) and low density lipoprotein (LDL), as well as a water soluble substrate, p-nitrophenyl butyrate (PNPB). There was no reaction with rHDL and LDL, but 30% of the activity with PNPB was retained. This suggests that the deleted region plays a role in interfacial binding, while the active site core is not disrupted. We thus conclude that this region (C50-C74) forms part of the interfacial binding domain of LCAT.
Adimoolam
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Department of Biochemistry, University of Illinois, College of Medicine at Urbana-Champaign 61801, USA.
Jonas
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A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D008049:Lipase / Q000235:genetics; D008075:Lipoproteins, HDL / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D008969:Molecular Sequence Data; D017384:Sequence Deletion; D002785:Sterol O-Acyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D018076:DNA, Complementary; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D002785:Sterol O-Acyltransferase; D008049:Lipase