Kinetic mechanism of human histone acetyltransferase P/CAF

Human transcriptional coactivator P/CAF (p300/CBP-associating factor) is a histone acetyltransferase (HAT) and is a member of the GNAT (GCN5 related N -acetyltransferases) superfamily. P/CAF was originally identified by its ab ility to activate transcription of a variety of genes through its interacti on with p300/CBP, Though Lys-14 of histone H3 appears to be the preferred s ubstrate, other nonhistone proteins can also serve as substrates for P/CAF. However, few studies have addressed the catalytic and kinetic mechanisms o f histone/protein acetylation by P/CAF. In this study, we have systematical ly determined the kinetic mechanism for P/CAF, identified the critical ioni zations for binding/catalysis, and established the rate-limiting step in tu rnover. This was accomplished by a variety of approaches including FH-depen dent activity measurements, Bi-substrate kinetic analysis, authentic produc t inhibition by coenzyme A (CoA) and acetylated H3 (Ac-Lys-14) peptide, dir ect measurements of substrate/product binding affinities (equilibrium dialy sis), and a pre-steady-state quench-flow analysis. The results are consiste nt with a fully ordered Bi-Bi kinetic mechanism, where chemical catalysis i s rate-determining Acetyl-CoA (AcCoA) binds with high affinity (K-d = 0.64 +/- 0.12 mu M) to the free form of the enzyme. Histone H3 peptide binds (ap parent K-d = 116 +/- 17 mu M) only after AcCoA is bound. No H3 peptide bind ing to the free enzyme was detectable, In the ternary complex, the epsilon- amino of Lys-14 (H3 peptide substrate) directly attacks the carbonyl carbon of AcCoA, transferring the acetyl group to the acceptor peptide substrate (rate-limiting step). Products are released in an ordered fashion, with Ac- Lys-ld, H3 released first followed by release of CoA, The pH dependency of the k(cat)/K-m parameter revealed two P/CAF ionizable groups (pK(a) values of 6.9 and 7.5) that must be unprotonated for activity. The group with a pK (a) value 7.5 was assigned to Glu-570, which is the proposed general base c atalyst, abstracting a proton from the epsilon-amino group and facilitating nucleophilic attack.