Transcriptional coactivator protein p300 - Kinetic characterization of itshistone acetyltransferase activity

The p300/cAMP response element-binding protein-binding protein (CBP) family members include human p300 and cAMP response element-binding protein-bindi ng protein, which are both important transcriptional coactivators and histo ne acetyltransferases. Although the role of these enzymes in transcriptiona l regulation has been extensively documented, the molecular mechanisms of p 300 and CBP histone acetyltransferase catalysis are poorly understood. Here in, we describe the first detailed kinetic characterization of p300 using f ull-length purified recombinant enzyme. These studies have employed peptide substrates to systematically examine the substrate specificity requirement s and the kinetic mechanism of this enzyme. The importance of nearby positi vely charged residues in lysine targeting was demonstrated. The strict stru ctural requirement of the lysine side chain was shown. The catalytic mechan ism of p300 was shown to follow a ping-pong kinetic pathway and viscosity e xperiments revealed that product release and/or a conformational change wer e likely rate-limiting in catalysis. Detailed analysis of the p300 selectiv e inhibitor Lys-CoA showed that it exhibited slow, tight-binding kinetics.