The transcriptional coactivator GCN5 from yeast (yGCN5) is a histone acetyltransferase that is essential for activation of target genes. GCN5 is a member of a large family of histone acetyltransferases that are conserved between yeast and humans. To understand the molecular mechanisms of histone/protein acetylation, a detailed kinetic analysis was performed. Bi-substrate kinetic analysis using acetyl-coenzyme A (AcCoA) and an H3 histone synthetic peptide indicated that both substrates must bind to form a ternary complex before catalysis. Product inhibition studies revealed that the product CoA was a competitive inhibitor versus AcCoA. Desulfo-CoA, a dead-end inhibitor, also demonstrated simple competitive inhibition versus AcCoA. Acetylated (Lys14Ac) H3 peptide displayed noncompetitive inhibition against both H3 peptide and AcCoA. These results support a sequential ternary complex (ordered Bi-Bi) kinetic mechanism, where AcCoA binds first, followed by H3 histone. Acetylated (Lys14Ac) H3 product is released first, and CoA is the last product to leave. Also, two methods were developed to measure the binding affinities of AcCoA/CoA for GCN5. Employing the fluorescent CoA analog etheno-CoA (epsilonCoA, 1-N(6)-etheno-CoA), a K(d) for epsilonCoA of 5.1 +/- 1.1 microm was determined by fluorescence anisotropy. This value was similar to the K(d) value of 8.5 +/- 2.6 microm for AcCoA obtained using equilibrium dialysis and to the K(i) (inhibition constant) of 6.7 microm for CoA obtained from steady-state kinetic assays. Together, these data suggest that the acetyl moiety of AcCoA contributes little to the binding energy.