title: Acetyl-lysine analog peptides as mechanistic probes of protein deacetylases.
abstract: Class III histone deacetylases (Sir2 or sirtuins) catalyze the NAD+-dependent conversion of acetyl-lysine residues to nicotinamide, 2'-O-acetyl-ADP-ribose (OAADPr), and deacetylated lysine. Class I and II HDACs utilize a different deacetylation mechanism, utilizing an active site zinc to direct hydrolysis of acetyl-lysine residues to lysine and acetate. Here, using ten acetyl-lysine analog peptides, we have probed the substrate binding pockets of sirtuins and investigated the catalytic differences among sirtuins and class I and II deacetylases. For the sirtuin Hst2, acetyl-lysine analog peptide binding correlated with the hydrophobic substituent parameter pi with a slope of -0.35 from a plot of log Kd versus pi. Interestingly, propionyl- and butyryl-lysine peptides were found to bind tighter to Hst2 compared with acetyl-lysine peptide and showed measurable rates of catalysis with Hst2, Sirt1, Sirt2, and Sirt3, suggesting propionyl- and butyryl-lysine proteins may be sirtuin substrates in vivo. Unique among the acetyl-lysine analog peptides examined, homocitrulline peptide produced ADP-ribose instead of the corresponding OAADPr analog. The electron-withdrawing nature of each acetyl analog had a profound impact on the deacylation rate between deacetylase classes. The rate of catalysis with the acetyl-lysine analog peptides varied over five orders of magnitude with the class III deacetylase Hst2, revealing a linear free energy relationship with a slope of -1.57 when plotted versus the Taft constant, sigma*. HDAC8, a class I deacetylase, displayed the opposite trend with a slope of +0.79. These results are applicable toward the development of selective substrates and other mechanistic probes of protein deacetylases.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706-1532, USA.
authors.forename: Brian C
authors.initials: BC
authors.lastname: Smith
authors.forename: John M
authors.initials: JM
authors.lastname: Denu
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D006655:Histone Deacetylases / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D008239:Lysine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism; D009536:Niacinamide / Q000378:metabolism; D038961:O-Acetyl-ADP-Ribose / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D037761:Sirtuins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C449679:2-O-acetyl-ADP-ribose; D015335:Molecular Probes; D038961:O-Acetyl-ADP-Ribose; D010455:Peptides; D009536:Niacinamide; D037761:Sirtuins; D006655:Histone Deacetylases; D008239:Lysine