Mutational analysis of conserved residues in the GCN5 family of histone acetyltransferases.

GCN5 is a critical transcriptional co-activator and is the defining member of a large superfamily of N-acetyltransferases. GCN5 catalyzes the transfer of an acetyl group from acetyl-CoA to the epsilon-amino of lysine 14 within the core H3 histone protein. Previous biochemical analyses have indicated a fully ordered kinetic mechanism. Recent structural studies have implicated several conserved residues in catalysis and substrate binding. Here the roles of Glu-173, His-145, and Asp-214 in yeast GCN5 have been evaluated using site-directed mutagenesis, steady state and pre-steady state kinetics, pH analysis, isotope partitioning, and equilibrium binding studies. The results with wild type and E173Q, H145A, and D214A mutants are consistent with chemical catalysis being rate-determining in turnover. All mutants exhibited K(d) values (3.5-8.5 microm) for AcCoA that were similar to wild type enzyme, indicating no functional role for these residues in AcCoA binding. The E173Q mutant demonstrated a approximately 500-600-fold decreases in k(cat) and k(cat)/K(m),(H3), consistent with Glu-173 acting as the general base catalyst as proposed previously. No significant effect was observed on substrate binding steps. His-145 was identified as a residue in the peptide binding cleft that must be unprotonated (pK(a) = 5.8) for peptide binding and likely hydrogen-bonds to the Ser-10 hydroxyl of histone H3. His-145 also contributes to lowering the pK(a) value (by 0.8 units) of general base Glu-173 through a water-mediated hydrogen bond to the carboxylate side chain. Analysis of D214A revealed an obligate protein isomerization step that occurs after AcCoA binding and permits efficient peptide binding. Asp-214 is part of a conformationally flexible loop that mediates the isomerization by stabilizing distinct conformers of the protein.