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Identification of crucial residues involved in the catalysis of acyl-coenzyme A:cholesterol acyltransferase.

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Call No.	QP606.A33 L8 2002
Author	Lu, Xiaohui
Title	Identification of crucial residues involved in the catalysis of acyl-coenzyme A:cholesterol acyltransferase.
Date	2002
Abstract	Acyl-coenzyme A: cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis and in early stages of atherogenesis. ACAT has been investigated as a pharmaceutical target, yet its active site(s) remain largely unknown. Earlier studies showed that ACAT activity was inhibited by cysteine-specific modification reagents, suggesting that certain cysteines may be near ACAT's active site. In the current study, I utilized a site-directed mutagenesis approach with single substitution to replace cysteine with alanine. The results showed that all mutants remained partially active. I next constructed ACAT1 mutants with decreasing numbers of cysteines. All of these mutants also retained partial ACAT activity either expressed in insect cells or in ACAT-deficient CHO cells. These data indicate that none of the cysteines is essential for ACAT catalysis. I also found that ACAT1 containing only one cysteine at amino acid 467 remained sensitive to pCMBS inhibition, suggesting that the C467 is near the active site. The cysteine-free ACAT-1 mutant preserves ACAT-1 catalytic properties. It will be useful in ACAT-1 structural and functional studies. To identify crucial residues in ACAT1 catalysis, I replaced each of the sixteen conserved polar amino acids in ACAT1 with alanine or other residues. My results showed that 5269, a proposed active center residue, is not essential for ACAT1 catalysis. I also showed that point mutations in any of the six residues K266, D400, N421, S456, H460, and E461 caused a severe loss in ACAT1 activity. H460 is an invariant residue within a membrane-bound O-acyltransferases superfamily. It is located within a long hydrophobic region. These data suggest that H460, as well as K266, D400, N421, S456, and E461, may constitute as part of the ACAT active site. I also discovered that ACAT1 contains oleoyl-CoA hydrolase activity. Further analysis showed that among H460A, S456A and D400A ACAT1 mutants, only the D400A mutant lost more than 70% of the oleoyl-CoA hydrolase activity, suggesting that the ACAT1 active center might consist of two different sets of functional groups.
Description	1 online resource (ix, 157 leaves) : illustrations (some color)
Format	application/pdf
Advisor	Barlowe, Charles K.
Subject	Acyltransferases. ; Cholesterol -- Metabolism. ; Catalysis. ; Acyl Coenzyme A. ; Cholesterol Acyltransferase.
College	Dartmouth Medical School -- Department of Biochemistry.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D)--Dartmouth College.
Copyright	In Copyright. This material is protected by copyright and/or related rights. You are free to use this material in any way that is permitted by copyright law that applies to your use. For other uses, you need to obtain permission from the rights-holder(s). http://rightsstatements.org/page/InC/1.0/
DOI	https://doi.org/10.1349/ddlp.2805

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Identification of crucial residues involved in the catalysis of acyl-coenzyme A:cholesterol acyltransferase.

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