The enzyme enoyl-ACP reductase from E. coli (also called FabI) is a well-studied enzyme that is the target of the antibacterial drug triclosan. The native structure of E. coli enoyl-ACP reductase is a homotetramer, which suggests the possibility of allosterism, but no allosteric effects have been previously reported for this enzyme. In this study, we used native MS to measure the affinity of the coenzymes NADH and NAD+ for the binding site in each monomer of the E. coli enoyl-ACP reductase homotetramer. We performed all MS experiments using nanospray ESI with a Waters Synapt G2 Si QTOF mass spectrometer. By comparing the measured affinities to the expected values calculated from the normal statistical relationship between affinities at successive binding sites, we observed a homotropic allosteric effect for each coenzyme. Treatment of the enzyme with the antibacterial drug triclosan caused a ~100-fold increase in affinity of FabI for NAD+ which is generally consistent with affinity measurements from solution-phase experiments. Additionally, we used HD exchange to demonstrate that triclosan treatment induced a conformational change in the enzyme and MS/MS experiments that showed the formation of a highly stable interaction between the enzyme and NAD+. These results are generally consistent with the extensive body of literature describing this enzyme, but they also provide new insights into the protein-ligand interactions of enoyl-ACP reductase with its coenzymes and the inhibitor triclosan.

Using native MS to identify homotropic allosteric effects of the coenzymes of enoyl-ACP reductase from E. coli (FabI)

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.