Identification of putative active site residues of ACAT enzymes.
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.
Das
Akash
A
Department of Biochemistry, Section on Lipid Sciences, Wake Forest University School of Medicine, Winston Salem, NC 27157-1040, USA.
Davis
Matthew A
MA
Rudel
Lawrence L
LL
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D016466:CHO Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017510:Protein Folding; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry; D002785:Sterol O-Acyltransferase / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry; D000097587:Sterol O-Acyltransferase 2
D001224:Aspartic Acid; D006639:Histidine; D012694:Serine; D002785:Sterol O-Acyltransferase; D014443:Tyrosine; D000097587:Sterol O-Acyltransferase 2; C493492:sterol O-acyltransferase 1