Dissecting the Effects of Resveratrol on SIRT1 Activity Against Different Peptide Substrates

Abstract

The enzyme SIRT1 is a member of a class of Sirtuins, which are NAD+-dependent protein deacylases that are involved in the metabolic and aging processes of cells. The activity of SIRT1 can be allosterically modulated by compounds known as Sirtuin Activating Compounds (STACs), such as resveratrol, which bind to the STAC Binding Domain (SBD) of SIRT1 in the N-terminus. End-point activity assays have shown that resveratrol can have different effects on SIRT1 activity depending on the acetylated peptide substrate used, including an increase, decrease, or no change in deacetylation activity. The goal of our project is to assess the effect of resveratrol on the catalytic rate (kcat) and substrate recognition (KM) of SIRT1 against different acetylated peptide substrates, rather than only examining the overall catalytic efficiency. A large change in the KM of SIRT1 with a select peptide would suggest that resveratrol had an effect on the substrate recognition of the enzyme, whereas a change in the kcat would imply that resveratrol caused an alteration in the catalytic rate of the enzyme for that specific peptide. We hypothesize that resveratrol allosterically affects SIRT1 activity by changing the conformation of the SBD region, which would likely result in a large change in the KM, such as a two-fold change in KM or higher. To test this hypothesis, enzyme-coupled assays involving SIRT1 acting on the peptide substrates Ac-CSNK, Ac-p300, Ac-H4, and Ac-p53 with and without resveratrol have been performed. Previous end-point assay studies have shown that resveratrol decreases SIRT1 deacetylation activity against Ac-CSNK as well as Ac-p300, but increases SIRT1 deacetylation activity against Ac-H4, and produces no change in deacetylation activity against the substrate Ac-p53. The Michaelis-Menten parameters for SIRT1 activity with and without resveratrol were compared, allowing observation of the effects of resveratrol on SIRT1's KM and kcat values. Understanding how resveratrol affects SIRT1 deacetylation activity will provide further insight into the development of drugs that can alter SIRT1 activity in a specific way to mitigate the effects of harmful diseases in humans.