title: Catalytic mechanism of a MYST family histone acetyltransferase.
abstract: Distinct catalytic mechanisms have been proposed for the Gcn5 and MYST histone acetyltransferase (HAT) families. Gcn5-like HATs utilize an ordered sequential mechanism involving direct nucleophilic attack of the N-epsilon-lysine on the enzyme-bound acetyl-CoA. Recently, MYST enzymes were reported to employ a ping-pong route of catalysis via an acetyl-cysteine intermediate. Here, using the prototypical MYST family member Esa1, and its physiological complex (piccolo NuA4), steady-state kinetic analyses revealed a kinetic mechanism that requires the formation of a ternary complex prior to catalysis, where acetyl-CoA binds first and CoA is the last product released. In the absence of histone acceptor, slow rates of enzyme auto-acetylation (7 x 10(-4) s(-1), or approximately 2500-fold slower than histone acetylation; kcat = 1.6 s(-1)) and of CoA formation (0.0021 s(-1)) were inconsistent with a kinetically competent acetyl-enzyme intermediate. Previously, Cys-304 of Esa1 was the proposed nucleophile that forms an acetyl-cysteine intermediate. Here, mutation of this cysteine (C304A) in Esa1 or within the piccolo NuA4 complex yielded an enzyme that was catalytically indistinguishable from the wild type. Similarly, a pH rate (kcat) analysis of the wild type and C304A revealed an ionization (pKa = 7.6-7.8) that must be unprotonated. Mutation of a conserved active-site glutamate (E338Q) reduced kcat approximately 200-fold at pH 7.5; however, at higher pH, E338Q exhibited nearly wild-type activity. These data are consistent with Glu-338 (general base) activating the N-epsilon-lysine by deprotonation. Together, the results suggest that MYST family HATs utilize a direct-attack mechanism within an Esa1 x acetyl-CoA x histone ternary complex.
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Berndsen
authors.forename: Brittany N
authors.initials: BN
authors.lastname: Albaugh
authors.forename: Song
authors.initials: S
authors.lastname: Tan
authors.forename: John M
authors.initials: JM
authors.lastname: Denu
mesh_terms: D000123:Acetyltransferases; D002384:Catalysis; D051548:Histone Acetyltransferases / Q000378:metabolism*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D000123:Acetyltransferases; C469780:Yng2 protein, S cerevisiae; C496551:Esa1 protein, S cerevisiae; D051548:Histone Acetyltransferases; C504740:NuA4 protein, S cerevisiae