Affinity labeling fatty acyl-CoA synthetase with 9-p-azidophenoxy nonanoic acid and the identification of the fatty acid-binding site

Paul N Black, Concetta C DiRusso, David Sherin, Robert MacColl, Jens Knudsen, James D. Weimar

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP-forming, EC 6.2.1.3) catalyzes the esterification of fatty acids to CoA thioesters for further metabolism and is hypothesized to play a pivotal role in the coupled transport and activation of exogenous long-chain fatty acids in Escherichia coli. Previous work on the bacterial enzyme identified a highly conserved region (FACS signature motif) common to long- and medium-chain acyl-CoA synthetases, which appears to contribute to the fatty acid binding pocket. In an effort to further define the fatty acid-binding domain within this enzyme, we employed the affinity labeled long-chain fatty acid [3H]9-p-azidophenoxy nonanoic acid (APNA) to specifically modify the E. coli FACS. [3H]APNA labeling of the purified enzyme was saturable and specific for long-chain fatty acids as shown by the inhibition of modification with increasing concentrations of palmitate. The site of APNA modification was identified by digestion of [3H]APNA cross-linked FACS with trypsin and separation and purification of the resultant peptides using reverse phase high performance liquid chromatography. One specific 3H-labeled peptide, T33, was identified and following purification subjected to NH2-terminal sequence analysis. This approach yielded the peptide sequence PDATDEIIK, which corresponded to residues 422 to 430 of FACS. This peptide is immediately adjacent to the region of the enzyme that contains the FACS signature motif (residues 431-455). This work represents the first direct identification of the carboxyl-containing substrate-binding domain within the adenylate-forming family of enzymes. The structural model for the E. coli FACS predicts this motif lies within a cleft separating two distinct domains of the enzyme and is adjacent to a region that contains the AMP/ATP signature motif, which together are likely to represent the catalytic core of the enzyme.

Original languageEnglish (US)
Pages (from-to)38547-38553
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number49
DOIs
StatePublished - Dec 8 2000

Fingerprint

Coenzyme A Ligases
Acyl Coenzyme A
Labeling
Fatty Acids
Binding Sites
Enzymes
Escherichia coli
Peptides
Adenosine Monophosphate
Coenzyme A
Purification
Esterification
Palmitates
Structural Models
High performance liquid chromatography
Reverse-Phase Chromatography
Ligases
pelargonic acid
Metabolism
Trypsin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Affinity labeling fatty acyl-CoA synthetase with 9-p-azidophenoxy nonanoic acid and the identification of the fatty acid-binding site. / Black, Paul N; DiRusso, Concetta C; Sherin, David; MacColl, Robert; Knudsen, Jens; Weimar, James D.

In: Journal of Biological Chemistry, Vol. 275, No. 49, 08.12.2000, p. 38547-38553.

Research output: Contribution to journalArticle

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abstract = "Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP-forming, EC 6.2.1.3) catalyzes the esterification of fatty acids to CoA thioesters for further metabolism and is hypothesized to play a pivotal role in the coupled transport and activation of exogenous long-chain fatty acids in Escherichia coli. Previous work on the bacterial enzyme identified a highly conserved region (FACS signature motif) common to long- and medium-chain acyl-CoA synthetases, which appears to contribute to the fatty acid binding pocket. In an effort to further define the fatty acid-binding domain within this enzyme, we employed the affinity labeled long-chain fatty acid [3H]9-p-azidophenoxy nonanoic acid (APNA) to specifically modify the E. coli FACS. [3H]APNA labeling of the purified enzyme was saturable and specific for long-chain fatty acids as shown by the inhibition of modification with increasing concentrations of palmitate. The site of APNA modification was identified by digestion of [3H]APNA cross-linked FACS with trypsin and separation and purification of the resultant peptides using reverse phase high performance liquid chromatography. One specific 3H-labeled peptide, T33, was identified and following purification subjected to NH2-terminal sequence analysis. This approach yielded the peptide sequence PDATDEIIK, which corresponded to residues 422 to 430 of FACS. This peptide is immediately adjacent to the region of the enzyme that contains the FACS signature motif (residues 431-455). This work represents the first direct identification of the carboxyl-containing substrate-binding domain within the adenylate-forming family of enzymes. The structural model for the E. coli FACS predicts this motif lies within a cleft separating two distinct domains of the enzyme and is adjacent to a region that contains the AMP/ATP signature motif, which together are likely to represent the catalytic core of the enzyme.",
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