Marek Cebrat

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p300/CBP-associated factor histone acetyltransferase processing of a peptide substrate. Kinetic analysis of the catalytic mechanism.

O D Lau; A D Courtney; A Vassilev; L A Marzilli; R J Cotter; Y Nakatani; P A Cole (Profiled Authors: Robert Cotter; Philip Cole)

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
The Journal of biological chemistry 2000;275(29):21953-9.

p300/CBP-associated factor (PCAF) is a histone acetyltransferase that plays an important role in the remodeling of chromatin and the regulation of gene expression. It has been shown to catalyze preferentially acetylation of the epsilon-amino group of lysine 14 in histone H3. In this study, the kinetic mechanism of PCAF was evaluated with a 20-amino acid peptide substrate derived from the amino terminus of histone H3 (H3-20) and recombinant bacterially expressed PCAF catalytic domain (PCAF(cat)). The enzymologic behavior of full-length PCAF and PCAF(cat) were shown to be similar. PCAF-catalyzed acetylation of the substrate H3-20 was shown to be specific for Lys-14, analogous to its behavior with the full-length histone H3 protein. Two-substrate kinetic analysis displayed an intersecting line pattern, consistent with a ternary complex mechanism for PCAF. The dead-end inhibitor analog desulfo-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 uncompetitive versus acetyl-CoA. The potent bisubstrate analog inhibitor H3-CoA-20 was competitive versus acetyl-CoA and noncompetitive versus H3-20. Taken together, these inhibition patterns support an ordered BiBi kinetic mechanism for PCAF in which acetyl-CoA binding precedes H3-20 binding. Viscosity experiments suggest that diffusional release of product is not rate-determining for PCAF catalysis. These results provide a mechanistic framework for understanding the detailed catalytic behavior of an important subset of the histone acetyltransferases and have significant implications for molecular regulation of and inhibitor design for these enzymes.

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