Identification of putative active site residues of acyl-Coenzyme A:cholesterol acyltransferase enzymes

Akash Das, Matthew A. Davis, and Lawrence L. Rudel

Pathology, Wake Forest Univ. Health Sciences, Winston-Salem, NC 27157

Corresponding Author: 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 proteins was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. As 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 towards serine modifying reagents whereas ACAT1 was not. Further studies indicated tyrosine residues may be important for ACAT activity. Mutational analysis showed the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity while 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.

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