Dissecting the Mechanism of Allosteric Regulation of SIRT1

The enzyme SIRT1 is a lysine deacetylase that plays a part in various pathways in the body, including insulin secretion and neurodegeneration, rendering it as a potential drug target for common diseases such as Type II diabetes and Alzheimer's disease. The activity of SIRT1 can be modulated allosterically by 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 dissect the activity of SIRT1 with and without resveratrol against different acetylated peptide substrates. Rather than only examining the overall catalytic efficiency, our goal is to assess the effect of resveratrol on the catalytic rate (kcat ) and substrate recognition (KM ) of SIRT1 against different acetylated peptide substrates. Upon addition of resveratrol, a large change in the KM of SIRT1 with a select peptide would imply that resveratrol had an effect on the substrate recognition of the enzyme, whereas a large change in the kcat may indicate that resveratrol caused an alteration in the catalytic rate of the enzyme for that specific peptide. We hypothesize that resveratrol allosterically alters SIRT1 activity by changing the conformation of the SBD region relative to the catalytic core, which would likely result in a large change in the KM , such as a two-fold change in KM or higher, indicating a change in the efficiency of SIRT1 substrate recognition. To test this hypothesis, enzyme-coupled assays involving SIRT1 acting on the peptide substrates 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-p300, but increases activity against Ac-H4, and does not affect SIRT1 activity against 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. © FASEB.