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Journal of Lipid Research, Vol. 49, 1770-1781, August 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Identification of putative active site residues of ACAT enzymes^*

Akash Das^*, Matthew A. Davis and Lawrence L. Rudel^1,*,

^* Department of Biochemistry, Section on Lipid Sciences, Wake Forest University School of Medicine, Winston Salem, NC 27157-1040
Department of Pathology, Section on Lipid Sciences, Wake Forest University School of Medicine, Winston Salem, NC 27157-1040

^* This work was supported by National Institutes of Health Grant P01-HL-49373 to L.L.R.

Published, JLR Papers in Press, May 13, 2008.

¹ To whom correspondence should be addressed. e-mail: lrudel{at}wfubmc.edu

In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

Supplementary key words ACAT • cholesterol • cholesteryl ester • catalytic triad

Abbreviations: AGM, African green monkey; BSS, balanced salt solution; CE, cholesteryl ester; DSG, disuccinimidyl glutarate; DEPC, diethylpyrocarbonate; HDSF, hexadecylsulfonylfluoride; ICL, iodine monochloride; IAA, iodoacetamide; NP-LLL-VS, 4-hydroxy-3-nitrophenylacetyl-Leu-Leu-Leu-vinylsulfone; PNS: postnuclear supernatant; WT, wild type

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