Publication Date

Summer 2020

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Advisor

Ningkun Wang

Keywords

Allostery, Kinetics, Resveratrol, SAXS, SIRT1, Sirtuin

Subject Areas

Biochemistry

Abstract

SIRT1 is an NAD+ dependent deacetylase that has been implicated in many important cellular functions such as neurodegeneration and aging. SIRT1 has also been studied with resveratrol which was shown to modulate activity. While the mechanism of SIRT1 has been studied extensively, exact details are still not known, specifically the allosteric regulation by the N-terminal domain. We wanted to see if there is a correlation between the activity of SIRT1 and the distance of the N-terminal domain relative to the catalytic core. First we examined the kinetic parameters (kcat and KM) of SIRT1 with peptide substrates with and without resveratrol. We used a continuous coupled enzyme kinetics assay to see if the main contributors to the different activity of SIRT1 were either KM or kcat. We found that KM is the main contributor, suggesting that resveratrol affects SIRT1 activity by a change in substrate recognition, likely by altering SIRT1’s conformation. We also determined the overall conformational change of SIRT1 using small angle X-ray scattering (SAXS) techniques. We examined the conformation of SIRT1 complexed with different combinations of resveratrol and peptide substrates. Current SAXS profiles showed that resveratrol alters the overall conformation of SIRT1; however more experiments are needed to confirm whether there is a correlation between these conformational effects and resveratrol’s effect on SIRT1 activity. Taken together, these studies will help us better understand the mechanism of SIRT1 activity regulation by small molecules, specifically in terms of conformational changes.

Download

Available for download on Monday, October 20, 2025

Share

COinS