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

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 fur ther metabolism and is hypothesized to play a pivotal role in the coupled t ransport and activation of exogenous long-chain fatty acids in Escherichia coli, Previous work on the bacterial enzyme identified a highly conserved r egion (FACS signature motif) common to long- and medium-chain acyl-CoA synt hetases, which appears to contribute to the fatty acid binding pocket. In a n effort to further define the fatty acid-binding domain within this enzyme , we employed the affinity labeled long-chain fatty acid [H-3]9-p-azidophen oxy nonanoic acid (APNA) to specifically modify the E. coli FAGS. [H-3]APNA labeling of the purified enzyme was saturable and specific for long-chain fatty acids as shown by the inhibition of modification with increasing conc entrations of palmitate, The site of APNA modification was identified by di gestion of [H-3]APNA cross-linked FAGS with trypsin and separation and puri fication of the resultant peptides using reverse phase high performance liq uid chromatography, One specific H-3-labeled peptide, T33, was identified a nd following purification subjected to NH2-terminal sequence analysis. This approach yielded the peptide sequence PDATDEIIK which corresponded to resi dues 422 to 430 of FAGS. This peptide is immediately adjacent to the region of the enzyme that contains the FAGS signature motif (residues 431-455), T his work represents the first direct identification of the carboxyl-contain ing substrate-binding domain within the adenylate-forming family of enzymes . The structural model for the E, coli FAGS predicts this moth 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.