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Substrate-Dependent Modulation of SIRT2 by a Fluorescent Probe, 1-Aminoanthracene
David Bi1, Jie Yang1, Jun Young Hong2
1Department of Molecular Biology, Rowan University School of Osteopathic Medicine, Stratford, New Jersey 08084, United States.
Biochemistry
|September 17, 2020
Summary
The fluorescent probe 1-aminoanthracene (AMA) binds to sirtuin isoform 2 (SIRT2) within its hydrophobic tunnel. AMA
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Sirtuin isoform 2 (SIRT2) is a deacetylase enzyme.
- SIRT2's active site features a hydrophobic tunnel for substrate binding.
- Understanding substrate interactions is key to modulating SIRT2 activity.
Purpose of the Study:
- To investigate the binding of 1-aminoanthracene (AMA) to SIRT2's hydrophobic tunnel.
- To characterize AMA's interaction with SIRT2 in a substrate-dependent manner.
- To explore how substrate acylation influences AMA binding and SIRT2 activity.
Main Methods:
- Fluorescence spectroscopy to measure AMA binding affinity (Kd).
- Enzyme inhibition assays to determine IC50 values.
- Structural analysis to elucidate binding site interactions.
Main Results:
- AMA exhibits enhanced fluorescence upon binding to SIRT2 (>10-fold).
- AMA's affinity for SIRT2 increases significantly (approx. 10-fold) in the presence of a decanoylated peptide substrate.
- AMA competitively inhibits SIRT2 demyristoylase activity (IC50 = 21 microM).
- AMA binding site stability varies with substrate acylation (decanoylated/4-oxononanoylated vs. acetylated).
Conclusions:
- AMA binding to SIRT2 is substrate-dependent and occurs within the hydrophobic tunnel.
- AMA's interaction reveals insights into how substrate structure affects SIRT2's active site.
- This study provides a foundation for developing specific SIRT2 modulators.
