Crystal structure, catalytic mechanism, and substrate specificity of the GCN5 histone acetyltransferase family

Nuclear histone N-acetyltransferases (HATs) are a key group of enzymes which catalyze the acetylation of lysine residues in nucleosomal histones and numerous transcription factors. Acetylation of these proteins impacts several events in chromatin remodeling including transcriptional activation, DNA replication, and gene silencing. The GCN5/PCAF family is the mostly widely studied class of HATs with homologues in all eukaryotic kingdoms of life. Yeast GCN5 (yGCN5) was one of the first nuclear HATs to be identified, and its enzymatic activity was shown to be essential in promoting transcriptional activation in vivo. The crystal structure of the HAT domain of yGCN5 has been solved to 1.9 Å resolution. The core fold of the domain, consisting of a four-stranded β-sheet sandwiched against an amphipathic α-helix, is conserved in other N-acetyltransferases. Analysis of alanine-scanning mutants and the structure has revealed a conserved glutamate, which is positioned within the active site to function as a catalytic base. Mutation of this glutamate to a glutamine results in a 360-fold decrease in kcat in vitro, while the mutant is as defective as a GCN5 knockout in in vivo growth and transcription in yeast. The position of the glutamate in yGCN5\u27s active site correlates with the position of potential catalytic bases in the other N-acetyltransferases, thereby suggesting a common catalytic mechanism. In addition to the structural and functional studies, a sensitive fluorometric HAT assay has been developed to investigate the kinetics of the GCN5/PCAF HAT family. Utilizing this assay, the substrate specificity of human PCAF was examined using peptides of three reported in vivo substrates, histones H3 and H4 and the transcription factor p53. An analysis of the kcat and Km values of differing length histone H3 peptides revealed that the determinants for PCAF substrate recognition lie within a 19-residue sequence of the histone H3 N-terminus. Furthermore, histone H3 is acetylated 650-fold and 5300-fold more efficiently than comparable length histone H4 and p53 peptides, respectively, thereby demonstrating that histone H3 is the preferred substrate of PCAF