p300/CBP-associated factor histone acetyltransferase processing of a peptide substrate

p300/CBP-associated factor (PCAF) is a histone acetyltransferase that; play s an important role in the remodeling of chromatin and the regulation of ge ne expression. It has been shown to catalyze preferentially acetylation of the epsilon-amino group of lysine 14 in histone H3, In this study, the kine tic mechanism of PCAF was evaluated with a 20-amino acid peptide substrate derived from the amino terminus of histone H3 (H3-20) and recombinant bacte rially expressed PCAF catalytic domain (PCAF(cat)). The enzymologic behavio r of full-length PCAF and PCAF(cat) were shown to be similar. PCAF-catalyze d acetylation of the substrate H3-20 was shown to be specific for Lys-14, a nalogous to its behavior with the full-length histone H3 protein. Two-subst rate kinetic analysis displayed an intersecting line pattern, consistent wi th a ternary complex mechanism for PCAF, The dead-end inhibitor analog desu lfo-CoA was competitive versus acetyl-CoA and noncompetitive versus H3-20, The dead-end analog inhibitor H3-20 K14A was competitive versus H3-20 and u ncompetitive versus acetylCoA The potent bisubstrate analog inhibitor H3-Co A-20 was competitive versus acetyl-CoA and noncompetitive versus H3-20. Tak en together, these inhibition patterns support an ordered BiBi kinetic mech anism for PCAF in which acetyl-CoA binding precedes H3-20 binding. Viscosit y experiments suggest that diffusional release of product is not rate-deter mining for PCAF catalysis, These results provide a mechanistic framework fo r understanding the detailed catalytic behavior of an important subset of t he histone acetyltransferases and have significant implications for molecul ar regulation of and inhibitor design for these enzymes.