The study investigates the molecular mechanism by which resveratrol activates human SIRT1, a NAD+-dependent protein deacetylase. Resveratrol, a polyphenol found in grapes, has been shown to increase SIRT1 activity and extend lifespan in various organisms. The research found that while resveratrol activates SIRT1, it does not activate other Sir2 homologs such as yeast Sir2 and human SIRT2. The activation of SIRT1 by resveratrol is dependent on the presence of a fluorophore covalently attached to the peptide substrate, specifically the p53-AMC or p53-R110 peptides. The fluorophore is necessary for the observed activation, as the absence of a fluorophore does not affect SIRT1 activity. Further studies using competition assays and structural modeling suggest that resveratrol binding to SIRT1 induces a conformational change that enhances the binding affinity of the fluorophore-containing peptides. This conformational change allows the coumarin group of the p53-AMC peptide to bind more tightly to the enzyme, leading to increased deacetylation of the substrate. The findings have implications for the use of resveratrol as an activator of Sir2 homologs in vivo and highlight the importance of the fluorophore in the activation mechanism.The study investigates the molecular mechanism by which resveratrol activates human SIRT1, a NAD+-dependent protein deacetylase. Resveratrol, a polyphenol found in grapes, has been shown to increase SIRT1 activity and extend lifespan in various organisms. The research found that while resveratrol activates SIRT1, it does not activate other Sir2 homologs such as yeast Sir2 and human SIRT2. The activation of SIRT1 by resveratrol is dependent on the presence of a fluorophore covalently attached to the peptide substrate, specifically the p53-AMC or p53-R110 peptides. The fluorophore is necessary for the observed activation, as the absence of a fluorophore does not affect SIRT1 activity. Further studies using competition assays and structural modeling suggest that resveratrol binding to SIRT1 induces a conformational change that enhances the binding affinity of the fluorophore-containing peptides. This conformational change allows the coumarin group of the p53-AMC peptide to bind more tightly to the enzyme, leading to increased deacetylation of the substrate. The findings have implications for the use of resveratrol as an activator of Sir2 homologs in vivo and highlight the importance of the fluorophore in the activation mechanism.