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.