Kinetic and mass spectrometric analysis of p300 histone acetyltransferase domain autoacetylation

Acetylation of proteins by p300 histone acetyltransferase plays a critical role in the regulation of gene expression. The prior discovery of an autoacetylated regulatory loop in the p300 histone acetyltransferase (HAT) domain prompted us to further explore the mechanisms of p300 autoacetylation. Here we have described a kinetic and mass spectrometric analysis of p300 HAT autoacetylation. The rate of p300 HAT autoacetylation was approximately fourth order with respect to p300 HAT domain concentration and thus appeared to be a highly cooperative process. By showing that a catalytically defective p300 HAT domain could be efficiently acetylated by active p300 HAT, we deduced that autoacetylation occurs primarily by an intermolecular mechanism. This was further confirmed using a semisynthetic biotinylated p300 HAT domain that could be physically separated from the catalytically defective p300 HAT by avidin affinity chromatography. Autoacetylation catalyzed by p300 HAT was approximately 1000-fold more efficient than PCAF (p300/CREB-binding protein-associated factor)-mediated acetylation of catalytically defective p300 HAT. Using a novel tandem mass spectrometric approach, it was found to be possible to observe up to 17 autoacetylation events within the intact p300 regulatory loop. Kinetic analysis of the site specificity of p300 autoacetylation reveals a class of rapid events followed by a slower set of modifications. Several of these rapid autoacetylation sites correlate with an acetyltransferase-activating function based on prior mutagenesis analysis.     

The catalytic mechanism of the ESA1 histone acetyltransferase involves a self-acetylated intermediate

Yeast ESA1 is a member of the MYST subfamily of histone acetyltransferases (HATs), which use acetyl-coenzyme A (CoA) to acetylate specific Lys residues within histones to regulate gene expression. The structure of an ESA1-CoA complex reveals structural similarity to the catalytic core of the GCN5/PCAF subfamily of HAT proteins. Here we report additional structural and functional studies on ESA1 that demonstrate that histone acetylation proceeds through an acetyl-cysteine enzyme intermediate. This Cys residue is strictly conserved within the MYST members, suggesting a common mode of catalysis by this HAT subfamily. However, this mode of catalysis differs dramatically from the GCN5/PCAF subfamily, which mediate direct nucleophilic attack of the acetyl-CoA cofactor by the enzyme-deprotonated substrate lysine of the histone. These results demonstrate that different HAT subfamilies can use distinct catalytic mechanisms, which have implications for their distinct biological roles and for the development of HAT-specific inhibitors.     

Kinetic mechanism of the reaction catalyzed by nuclear histone acetyltransferase from calf thymus

The kinetic mechanism for calf thymus histone acetyltransferase A has been determined from the initial velocity studies. The kinetic patterns at low substrate concentrations suggest that the reaction proceeds via two half-reactions as in a ping-pong pathway with the formation of an acetyl-enzyme intermediate. Such acetyl-enzyme has been isolated and found to be chemically competent. In addition, product inhibition patterns by coenzyme A are consistent with a hybrid ping-pong mechanism. These findings indicate that the acetyltransferase A from calf thymus has two separate and independent binding sites, one for each of the two substrates. Consequently, the mechanism constructed for the acetyltransferase A catalyzed reaction may be described as a double-displacement, two-site ping-pong mechanism.     

Processing mechanism and substrate selectivity of the core NuA4 histone acetyltransferase complex

Esa1, an essential MYST histone acetyltransferase found in the yeast piccolo NuA4 complex (picNuA4), is responsible for genome-wide histone H4 and histone H2A acetylation. picNuA4 uniquely catalyzes the rapid tetra-acetylation of nucleosomal H4, though the molecular determinants driving picNuA4 efficiency and specificity have not been defined. Here, we show through rapid substrate trapping experiments that picNuA4 utilizes a nonprocessive mechanism in which picNuA4 dissociates from the substrate after each acetylation event. Quantitative mass spectral analyses indicate that picNuA4 randomly acetylates free and nucleosomal H4, with a small preference for lysines 5, 8, and 12 over lysine 16. Using a series of 24 histone mutants of H4 and H2A, we investigated the parameters affecting catalytic efficiency. Most strikingly, removal of lysine residues did not substantially affect the ability of picNuA4 to acetylate remaining sites, and insertion of an additional lysine into the H4 tail led to rapid quintuple acetylation. Conversion of the native H2A tail to an H4-like sequence resulted in enhanced multisite acetylation. Collectively, the results suggest picNuA4's site selectivity is dictated by accessibility on the nucleosome surface, the relative proximity from the histone fold domain, and a preference for intervening glycine residues with a minimal (n + 2) spacing between lysines. Functionally distinct from other HAT families, the proposed model for picNuA4 represents a unique mechanism of substrate recognition and multisite acetylation.