title: KDAC8 with High Basal Velocity Is Not Activated by N-Acetylthioureas.
abstract: Lysine deacetylases (KDACs) are enzymes that reverse the post-translational modification of lysine acetylation. Recently, a series of N-acetylthioureas were synthesized and reported to enhance the activity of KDAC8 with a fluorogenic substrate. To determine if the activation was general, we synthesized three of the most potent N-acetylthioureas and measured their effect with peptide substrates and the fluorogenic substrate under multiple reaction conditions and utilizing two enzyme purification approaches. No activation was observed for any of the three N-acetylthioureas under any assayed conditions. Further characterization of KDAC8 kinetics with the fluorogenic substrate yielded a kcat/KM of 164 ± 17 in the absence of any N-acetylthioureas. This catalytic efficiency is comparable to or higher than that previously reported when KDAC8 was activated by the N-acetylthioureas, suggesting that the previously reported activation effect may be due to use of an enzyme preparation that contains a large fraction of inactive enzyme. Further characterization with a less active preparation and additional substrates leads us to conclude that N-acetylthioureas are not true activators of KDAC8 and only increase activity if the enzyme preparation is below the maximal basal activity.
authors.affiliation: Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, Louisiana, United States of America.
authors.forename: Tasha B
authors.initials: TB
authors.lastname: Toro
authors.affiliation: Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, Louisiana, United States of America.
authors.forename: Subramanya
authors.initials: S
authors.lastname: Pingali
authors.affiliation: Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, Louisiana, United States of America.
authors.forename: Thao P
authors.initials: TP
authors.lastname: Nguyen
authors.affiliation: Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, Louisiana, United States of America.
authors.forename: Destane S
authors.initials: DS
authors.lastname: Garrett
authors.affiliation: Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, Louisiana, United States of America.
authors.forename: Kyra A
authors.initials: KA
authors.lastname: Dodson
authors.affiliation: Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, Louisiana, United States of America.
authors.forename: Kyara A
authors.initials: KA
authors.lastname: Nichols
authors.affiliation: Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, Louisiana, United States of America.
authors.forename: Rashad A
authors.initials: RA
authors.lastname: Haynes
authors.affiliation: Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, Louisiana, United States of America.
authors.forename: Florastina
authors.initials: F
authors.lastname: Payton-Stewart
authors.affiliation: Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, Louisiana, United States of America.
authors.forename: Terry J
authors.initials: TJ
authors.lastname: Watt
mesh_terms: D057075:Enzyme Assays; D005450:Fluorescamine / Q000737:chemistry; D006655:Histone Deacetylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013890:Thiourea / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D012097:Repressor Proteins; D005450:Fluorescamine; C062177:acetylthiourea; C409470:HDAC8 protein, human; D006655:Histone Deacetylases; D013890:Thiourea