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

Sojin An, Kyung Hyun Cho, Woo Song Lee, Jie Oh Lee, Young Ki Paik, Tae Sook Jeong

Research output: Contribution to journalArticle

8 Citations (Scopus)

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 languageEnglish
Pages (from-to)2741-2749
Number of pages9
JournalFEBS Letters
Volume580
Issue number11
DOIs
Publication statusPublished - 2006 May 15

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Sterol O-Acyltransferase
Catalysis
Histidine
Substrates
Catalyst activity
Baculoviridae
Enzymes
Sequence Homology
Substrate Specificity
Alanine
Isoenzymes
Catalytic Domain
Cholesterol
Technology

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

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title = "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",
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.",
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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 journalArticle

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

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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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