A critical role for the histidine residues in the catalytic function of acyl-CoA:cholesterol acyltransferase catalysis: evidence for catalytic difference between ACAT1 and ACAT2.
To investigate a role for histidine residues in the expression of normal acyl-CoA:cholesterol acyltransferase (ACAT) activity, the histidine residues located at five different positions in two isoenzymes were substituted by alanine, based on the sequence homology between ACAT1 and ACAT2. Among the 10 mutants generated by baculovirus expression technology, H386A-ACAT1, H460A-ACAT1, H360A-ACAT2, and H399A-ACAT2 lost their enzymatic activity completely. A reduction in catalytic activity is unlikely to result from structural changes in the substrate-binding pocket, because their substrate-binding affinities were normal. However, the enzymatic activity of H386A-ACAT1 was restored to <37% of the level of the wild-type activity when cholesterol was replaced by 25-hydroxycholesterol as substrate. H527A-ACAT1 and H501A-ACAT2, termed carboxyl end mutants, exhibit activities of approximately 96% and approximately 75% of that of the wild-type. Interestingly, H425A-ACAT1 showed 59% of the wild-type activity, in contrast to its equivalent mutant, H399A-ACAT2. These results demonstrate that the histidine residues located at the active site are very crucial both for the catalytic activity of the enzyme and for distinguishing ACAT1 from ACAT2 with respect to enzyme catalysis and substrate specificity.
An
Sojin
S
National Research Laboratory of Lipid Metabolism and Atherosclerosis, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea.
Cho
Kyung-Hyun
KH
Lee
Woo Song
WS
Lee
Jie-Oh
JO
Paik
Young-Ki
YK
Jeong
Tae-Sook
TS
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002784:Cholesterol / Q000378:metabolism; D017124:Conserved Sequence; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D002785:Sterol O-Acyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology; D000097587:Sterol O-Acyltransferase 2
D002784:Cholesterol; D004791:Enzyme Inhibitors; D006639:Histidine; D011994:Recombinant Proteins; D002785:Sterol O-Acyltransferase; D000097587:Sterol O-Acyltransferase 2; C493492:sterol O-acyltransferase 1