p300/CBP-associated factor histone acetyltransferase processing of a peptide substrate. Kinetic analysis of the catalytic mechanism.
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.
Lau
O D
OD
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Courtney
A D
AD
Vassilev
A
A
Marzilli
L A
LA
Cotter
R J
RJ
Nakatani
Y
Y
Cole
P A
PA
D000123:Acetyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D051548:Histone Acetyltransferases; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D015534:Trans-Activators / Q000378:metabolism*
D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D000123:Acetyltransferases; D051548:Histone Acetyltransferases