Scheme 1, three competing sites on one substrate (H3).

<p>Scheme 1, three competing sites on one substrate (H3).</p

Second order rate constant for nonenzymatic acetylation.

<p>(A) Concentrations of acetylated H3K36 as a function of time fit to a pseudo-first order reaction ([acetyl-CoA] = 200 µM) with an apparent rate of 5.8±0.3×10⁻² h⁻¹. (B) k_obs for the nonenzymatic acetylation of K36 as a function of acetyl-CoA concentration resulting in an apparent rate constant of 4.1÷0.6×10⁻⁴ µM⁻¹ h⁻¹.</p

Steady-state parameters of H3 (wt) and H3K14ac for Gcn5-mediated acetylation (mean ± standard error).

<p>In cases where the Hill coefficient (<i>n</i>_H) is greater than one, (k_cat/K_1/2<i>ⁿ</i>^H)_(app) was also calculated. The units for (k_cat/K_m^nH)_(app) are µM^−<i>n</i>H min⁻¹ dividing by k_nE to determine (k_cat/K_1/2<i>ⁿ</i>^H)_(app)/k_nE (k_nE  = 6.83±0.89×10⁻⁶ µM⁻¹ min⁻¹) results in µM to the power of the -<i>n</i>_H+1 (µM^−nh+1−). For this reason we can not calculate a ΔΔG_(app.) for anything other than K14ac (<i>n</i>_H = 1, ΔΔG_(app.)  = −9.3±0.08 kcal mol⁻¹).</p

Determination of steady-state kinetic parameters for K14 acetylation by Gcn5.

<p>(A) k_cat  = 12.1±0.1 min⁻¹ and K_m  = 0.5±0.02 µM are determined when [Gcn5]  = 18 nM, [acetyl-CoA]  = 200 μM with titrating 13 different H3 concentrations. (B) k_cat  = 11.6±0.2 min⁻¹ and K_m  = 0.7±0.05 µM are determined when [Gcn5]  = 18 nM, [H3]  = 10 μM with titrating different acetyl-CoA concentrations.</p

Structure of H3 (blue highlight) in nucleosome, which is constructed from the PDB 1KX5 nucleosome structure [54].

<p>The red dots show the lysine locations that are reportedly acetylated in this study. The grey bar represents one H3 sequence and the white ovals show the relative lysine sites and arrows are the location of arginines that are the only locations digested by trypsin after chemical propionylation or acetylation.</p

Multiple views of Gcn5-mediated H3 acetylation kinetics from bottom-up MS analysis, when [H3] = 12 μM, [Gcn5] = 180 nM, and [acetyl-CoA] = 200 μM.

<p>(A) Changes of modifications on KSTGGKAPR: K_aSTGGK_aAPR (open circle), K_aSTGGK_pAPR (open square), K_pSTGGK_aAPR (open triangle), and K_pSTGGK_pAPR (solid reverse triangle). (B) Changes of modifications on KQLATKAAR: K_aQLATK_aAAR (solid circle), K_aQLATK_pAAR (solid square), K_pQLATK_aAAR (solid triangle), and K_pQLATK_pAAR (open reverse triangle). The data of (A) and (B) were directly obtained from MS SRM analysis. (C) Kinetics of fractions of acetylated K9 (solid circle), K14 (solid square), K18 (solid triangle), and K23 (open reverse triangle). (D) Kinetic of total acetylated lysine concentration on H3. The plots of (C) was generated from the calculation of (A) and (B). Apparently, K14 is the primary acetylation lysine by Gcn5 catalysis. While only the total or multiple acetylation is monitored, the acetylation amount from minor acetylation sites could be neglected, especially at short time points.</p

Detection parameters of tryptic peptides from Histone H3.

<p>Detection parameters of tryptic peptides from Histone H3.</p

Differences in Specificity and Selectivity Between CBP and p300 Acetylation of Histone H3 and H3/H4

Although p300 and CBP lysine acetyltransferases are often treated interchangeably, the inability of one enzyme to compensate for the loss of the other suggests unique roles for each. As these deficiencies coincide with aberrant levels of histone acetylation, we hypothesized that the key difference between p300 and CBP activity is differences in their specificity/selectivity for lysines within the histones. Utilizing a label-free, quantitative mass spectrometry based technique, we determined the kinetic parameters of both CBP and p300 at each lysine of H3 and H4, under conditions we would expect to encounter in the cell (either limiting acetyl-CoA or histone). Our results show that while p300 and CBP acetylate many common residues on H3 and H4, they do in fact possess very different specificities, and these specificities are dependent on whether histone or acetyl-CoA is limiting. Steady-state experiments with limiting H3 demonstrate that both CBP and p300 acetylate H3K14, H3K18, H3K23, with p300 having specificities up to 10¹⁰-fold higher than CBP. Utilizing tetramer as a substrate, both enzymes also acetylate H4K5, H4K8, H4K12, and H4K16. With limiting tetramer, CBP displays higher specificities, especially at H3K18, where CBP specificity is 10³²-fold higher than p300. With limiting acetyl-CoA, p300 has the highest specificity at H4K16, where specificity is 10¹⁸-fold higher than CBP. This discovery of unique specificity for targets of CBP- vs p300-mediated acetylation of histone lysine residues presents a new model for understanding their respective biological roles and possibly an opportunity for selective therapeutic intervention

Determination of steady-state parameters of H3/H4-Asf1 acetylation catalyzed by Rtt109-Vps75 for individual lysines.

<p>The detailed experimental conditions are described in the section “Steady-state kinetic assays for Rtt109-Vps75”. The error bar represents the standard error in v/E. Panels (A) and (B) show the Michaelis-Menten behaviors for H3K9 and H3K23, respectively, when titrating H3/H4-Asf1 in the presence of saturating acetyl-CoA. Panels (C) and (D) show the superposition of steady-state kinetics for H3K9 and H3K23, respectively, on different histone conformations (H3: blue triangle, H3/H4-Asf1: red circle, and H3/H4: green square). The apparent kinetic parameters are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118516#pone.0118516.t001" target="_blank">Table 1</a>. When H3/H4 is complexed with Asf1, no cooperativity is detected, and the specificity and selectivity of K9 and K23 acetylation increases.</p

Steady-state parameters from kinetic assays of acetyl-CoA titration (mean ± standard error).

<p>Steady-state parameters from kinetic assays of acetyl-CoA titration (mean ± standard error).</p