title: Kinetic mechanism of human histone acetyltransferase P/CAF.
abstract: Human transcriptional coactivator P/CAF (p300/CBP-associating factor) is a histone acetyltransferase (HAT) and is a member of the GNAT (GCN5 related N-acetyltransferases) superfamily. P/CAF was originally identified by its ability to activate transcription of a variety of genes through its interaction with p300/CBP. Though Lys-14 of histone H3 appears to be the preferred substrate, other nonhistone proteins can also serve as substrates for P/CAF. However, few studies have addressed the catalytic and kinetic mechanisms of histone/protein acetylation by P/CAF. In this study, we have systematically determined the kinetic mechanism for P/CAF, identified the critical ionizations for binding/catalysis, and established the rate-limiting step in turnover. This was accomplished by a variety of approaches including pH-dependent activity measurements, Bi-substrate kinetic analysis, authentic product inhibition by coenzyme A (CoA) and acetylated H3 (Ac-Lys-14) peptide, direct measurements of substrate/product binding affinities (equilibrium dialysis), and a pre-steady-state quench-flow analysis. The results are consistent with a fully ordered Bi-Bi kinetic mechanism, where chemical catalysis is rate-determining. Acetyl-CoA (AcCoA) binds with high affinity (K(d) = 0.64 +/- 0.12 microM) to the free form of the enzyme. Histone H3 peptide binds (apparent K(d) = 116 +/- 17 microM) only after AcCoA is bound. No H3 peptide binding to the free enzyme was detectable. In the ternary complex, the epsilon-amino of Lys-14 (H3 peptide substrate) directly attacks the carbonyl carbon of AcCoA, transferring the acetyl group to the acceptor peptide substrate (rate-limiting step). Products are released in an ordered fashion, with Ac-Lys-14 H3 released first followed by release of CoA. The pH dependency of the k(cat)/K(m) parameter revealed two P/CAF ionizable groups (pK(a) values of 6.9 and 7.5) that must be unprotonated for activity. The group with a pK(a) value 7.5 was assigned to Glu-570, which is the proposed general base catalyst, abstracting a proton from the epsilon-amino group and facilitating nucleophilic attack.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA.
authors.forename: K G
authors.initials: KG
authors.lastname: Tanner
authors.forename: M R
authors.initials: MR
authors.lastname: Langer
authors.forename: J M
authors.initials: JM
authors.lastname: Denu
mesh_terms: D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D051548:Histone Acetyltransferases; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
chemical_list: D000123:Acetyltransferases; D018797:Cell Cycle Proteins; D051548:Histone Acetyltransferases; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor