Using Enzyme Kinetics and Small Angle X‐ray Scattering to Understand the Allosteric Regulation of SIRT1

Abstract

SIRT1 is an NAD+ dependent deacetylase located in the nucleus and cytoplasm of the cell. Research has shown that SIRT1 has been implicated to be involved in many important cellular functions such as neurodegenerative diseases and aging. SIRT1 has also been studied with resveratrol, a polyphenol, which was shown to modulate SIRT's enzyme activity. While the mechanism of SIRT1 has been studied extensively, some exact details are still not known, specifically regarding the allosteric regulation by the N‐terminal domain. We hypothesize that there is a correlation between the activity of SIRT1 and the dynamics of the N‐terminal domain relative to the catalytic core. We are testing this hypothesis by enzyme kinetics and small angle X‐ray scattering (SAXS) methods, using resveratrol as a tool to vary SIRT1 activity. A recent study examined the activity of SIRT1 on peptides of different acetylation sites and found that resveratrol could either increase, decrease or not change SIRT1 activity. We chose peptides from each of these categories as substrates to produce a range of SIRT1 activity change upon resveratrol addition: increase, no change and decrease. First we examined the kinetic parameters (kcat and KM) of SIRT1 with these peptide substrates with and without resveratrol. We are using a continuous enzyme‐coupled assay, rather than an endpoint assay used in the previous study, to see if whether kcat or KM contributes more to the activity change. We also studied the conformational dynamics of SIRT1 in the presence of these peptides and resveratrol using SAXS. The SAXS data informed us about the folding state and overall conformation of the protein, where most changes in conformation would be due to large motions of the more dynamic N‐terminal domain. Combining the data from Michaelis‐Menten enzyme assays and SAXS, we hope to identify a correlation between the conformational dynamics of the N‐terminal domain and the enzyme activity of SIRT1. Learning more about the allosteric mechanism of SIRT1 could aid in the design of therapeutic targets in modulating SIRT1 activity in the cell.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.