Modulating acetyl-CoA binding in the GCN5 family of histone acetyltransferases

The histone acetyltransferase (HAT) GCN5 is the founding member for a family of chromatin remodeling enzymes. GCN5 is the catalytic subunit of a large multi-subunit complex that functions in the regulation of gene activation via acetylation of lysine residues within the N-terminal tails of core histone proteins. Using acetyl-CoA as a co-substrate, the high affinity binding of acetyl-CoA is a critical first step in the reaction. Here, we examine the biochemical and biological importance of a conserved hydroxyl-bearing residue in signature motif A. Interestingly, one major exception is the Saccharomyces cerevisiae GCN5, where an alanine (Ala(190)) is located in the corresponding position. In related GCN5 family structures, a hydroxyl-containing side chain residue is hydrogen-bonded to the alpha-phosphate oxygen of CoA. We demonstrate that this key hydrogen bond contributes approximately 10-fold to the binding affinity of GCN5 HATs for acetyl-CoA. Human p300/CBP-associating factor, human GCN5, and tetrahymena GCN5 displayed dissociation constants (K(d)) for acetyl-CoA of 0.64 +/- 0.12, 0.56 +/- 0.15, and 0.62 +/- 0.17 microm, respectively. In contrast, S. cerevisiae GCN5 displayed a K(d) of 8.5 microm. When Ala(190) was replaced with threonine, the A190T derivative yielded a K(d) value of 0.56 +/- 0.1 microm for acetyl-CoA, completely restoring the higher affinity binding seen with the GCN5 homologs that naturally harbor a threonine at this position. Detailed kinetic analyses revealed that the A190T derivative was otherwise catalytically indistinguishable from wild type GCN5. We also demonstrate that the A190T allele rescued the slow growth phenotype and the defect in HO transcription caused by a deletion of GCN5. Furthermore, the A190T allele supported wild type levels of transcriptionally targeted and global histone H3 acetylation. In each case, the A190T derivative behaved similarly to wild type GCN5, suggesting that the efficacy of HAT activity by GCN5 is not limited by the availability of nuclear acetyl-CoA pools.