Characterization of a cysteine-selective inhibitor of the bacterial fatty acid synthase enzyme enoyl-ACP reductase using mass spectrometry
Presentation Type
Poster
Presentation Type
Submission
Keywords
enzyme, inhibitor, cysteine, enoyl-ACP reductase, EOPE, enzyme kinetic assay, mass spectrometry
Abstract
Covalent modification of specific enzyme residues has been a standard tool for studying protein function for many years. The development of compounds with high selectivity for specific amino acids has provided scientists with molecular probes that can be used to elucidate various functional characteristics of a protein of interest. In this study, we used the enzyme enoyl-ACP reductase (also called FabI) from Escherichia coli as a model system to explore the role of cysteine modification in this important bacterial enzyme. We synthesized the highly selective electrophilic probe, (E)-N-ethyl-4-oxo-4-phenylbut-2-enamide (abbreviated EOPE), and conjugated it with enoyl-ACP reductase in order to determine the role of cysteine residues in the function of this enzyme. Surprisingly, the EOPE reagent exhibited nanomolar potency in enzyme kinetic assays despite the complete absence of any reports of cysteine residues playing any important roles in the activity of this enzyme. We used a variety of protein mass spectrometry methods, including intact protein and top-down mass spectrometry, to investigate the binding stoichiometry of the EOPE with the enzyme as well as the position of the modification in the enzyme. These results may provide new insights into the function of this enzyme and suggest new routes for antibacterial drug development.
Faculty Mentor
Dr. Matthew Joyner
Funding Source or Research Program
Summer Undergraduate Research Program, Undergraduate Research Fellowship
Location
Waves Cafeteria
Start Date
24-3-2023 2:00 PM
End Date
24-3-2023 4:00 PM
Characterization of a cysteine-selective inhibitor of the bacterial fatty acid synthase enzyme enoyl-ACP reductase using mass spectrometry
Waves Cafeteria
Covalent modification of specific enzyme residues has been a standard tool for studying protein function for many years. The development of compounds with high selectivity for specific amino acids has provided scientists with molecular probes that can be used to elucidate various functional characteristics of a protein of interest. In this study, we used the enzyme enoyl-ACP reductase (also called FabI) from Escherichia coli as a model system to explore the role of cysteine modification in this important bacterial enzyme. We synthesized the highly selective electrophilic probe, (E)-N-ethyl-4-oxo-4-phenylbut-2-enamide (abbreviated EOPE), and conjugated it with enoyl-ACP reductase in order to determine the role of cysteine residues in the function of this enzyme. Surprisingly, the EOPE reagent exhibited nanomolar potency in enzyme kinetic assays despite the complete absence of any reports of cysteine residues playing any important roles in the activity of this enzyme. We used a variety of protein mass spectrometry methods, including intact protein and top-down mass spectrometry, to investigate the binding stoichiometry of the EOPE with the enzyme as well as the position of the modification in the enzyme. These results may provide new insights into the function of this enzyme and suggest new routes for antibacterial drug development.