Abstract
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 ∼96% and ∼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.
| Original language | English |
|---|---|
| Pages (from-to) | 2741-2749 |
| Number of pages | 9 |
| Journal | FEBS Letters |
| Volume | 580 |
| Issue number | 11 |
| DOIs | |
| Publication status | Published - 2006 May 15 |
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All Science Journal Classification (ASJC) codes
- Biophysics
- Structural Biology
- Biochemistry
- Molecular Biology
- Genetics
- Cell Biology
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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. / An, Sojin; Cho, Kyung Hyun; Lee, Woo Song; Lee, Jie Oh; Paik, Young Ki; Jeong, Tae Sook.
In: FEBS Letters, Vol. 580, No. 11, 15.05.2006, p. 2741-2749.Research output: Contribution to journal › Article
TY - JOUR
T1 - A critical role for the histidine residues in the catalytic function of acyl-CoA:cholesterol acyltransferase catalysis
T2 - Evidence for catalytic difference between ACAT1 and ACAT2
AU - An, Sojin
AU - Cho, Kyung Hyun
AU - Lee, Woo Song
AU - Lee, Jie Oh
AU - Paik, Young Ki
AU - Jeong, Tae Sook
PY - 2006/5/15
Y1 - 2006/5/15
N2 - 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 ∼96% and ∼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.
AB - 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 ∼96% and ∼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.
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UR - http://www.scopus.com/inward/citedby.url?scp=33646178938&partnerID=8YFLogxK
U2 - 10.1016/j.febslet.2006.04.035
DO - 10.1016/j.febslet.2006.04.035
M3 - Article
C2 - 16647063
AN - SCOPUS:33646178938
VL - 580
SP - 2741
EP - 2749
JO - FEBS Letters
JF - FEBS Letters
SN - 0014-5793
IS - 11
ER -