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Updated: Apr 17, 2026
Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates
Published on: February 27, 2016
Propofol inhibits SIRT2 deacetylase through a conformation-specific, allosteric site
Brian P Weiser1, Roderic G Eckenhoff2
1From the Departments of Anesthesiology and Critical Care and Pharmacology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104.
Abstract:
meta-Azi-propofol (AziPm) is a photoactive analog of the general anesthetic propofol. We photolabeled a myelin-enriched fraction from rat brain with [(3)H]AziPm and identified the sirtuin deacetylase SIRT2 as a target of the anesthetic. AziPm photolabeled three SIRT2 residues (Tyr(139), Phe(190), and Met(206)) that are located in a single allosteric protein site, and propofol inhibited [(3)H]AziPm photolabeling of this site in myelin SIRT2. Structural modeling and in vitro experiments with recombinant human SIRT2 determined that propofol and [(3)H]AziPm only bind specifically and competitively to the enzyme when co-equilibrated with other substrates, which suggests that the anesthetic site is either created or stabilized in enzymatic conformations that are induced by substrate binding. In contrast to SIRT2, specific binding of [(3)H]AziPm or propofol to recombinant human SIRT1 was not observed. Residues that line the propofol binding site on SIRT2 contact the sirtuin co-substrate NAD(+) during enzymatic catalysis, and assays that measured SIRT2 deacetylation of acetylated α-tubulin revealed that propofol inhibits enzymatic function. We conclude that propofol inhibits the mammalian deacetylase SIRT2 through a conformation-specific, allosteric protein site that is unique from the previously described binding sites of other inhibitors. This suggests that propofol might influence cellular events that are regulated by protein acetylation state.
Insights
Propofol, a general anesthetic, inhibits the mammalian deacetylase SIRT2. This inhibition occurs via a unique allosteric site, potentially influencing cellular events regulated by protein acetylation.
Area of Science:
- Biochemistry
- Neuroscience
- Pharmacology
Background:
- Propofol is a widely used general anesthetic.
- The anesthetic's precise molecular targets and mechanisms remain incompletely understood.
- Sirtuins are a class of NAD+-dependent deacetylases involved in various cellular processes.
Purpose of the Study:
- To identify molecular targets of propofol using a photoaffinity analog.
- To characterize the binding site and inhibitory mechanism of propofol on its identified target.
- To investigate the functional consequences of propofol binding to SIRT2.
Main Methods:
- Photolabeling of rat brain myelin-enriched fraction with meta-Azi-propofol (AziPm).
- Identification of photolabeled proteins using mass spectrometry.
- Structural modeling and in vitro enzymatic assays with recombinant human SIRT2 and SIRT1.
- Deacetylation assays using acetylated α-tubulin as a substrate.
Main Results:
- [(3)H]AziPm photolabeled and identified SIRT2 as a direct target.
- Propofol competitively inhibited [(3)H]AziPm binding to SIRT2 at an allosteric site involving residues Tyr(139), Phe(190), and Met(206).
- Propofol binding and inhibition were dependent on co-incubation with substrates, suggesting induced binding site formation.
- Propofol inhibited SIRT2 deacetylation activity, affecting acetylated α-tubulin.
- No specific binding of propofol or AziPm to SIRT1 was observed.
Conclusions:
- Propofol inhibits the mammalian deacetylase SIRT2 through a unique, conformation-specific allosteric site.
- The binding site is distinct from those of other known inhibitors.
- Propofol's inhibition of SIRT2 suggests a potential role in modulating cellular events regulated by protein acetylation.
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