Table 1.
Detection parameters of tryptic peptides from Histone H3.
Figure 1.
Scheme 1, three competing sites on one substrate (H3).
Figure 2.
Structure of H3 (blue highlight) in nucleosome, which is constructed from the PDB 1KX5 nucleosome structure [54].
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.
Figure 3.
Sites acetylated on histone H3.
(A) Gcn5 mediated acetylation of H3 (12 µM H3, 200 µMacetyl-CoA) at 0.5 (light grey), 1 (dark grey), and 2 (black) hours. (B) Nonenzymatic acetylation of H3 (12 µM) by acetyl-CoA (200 µM) at 6 (white), 21.5 (grey), and 73 (black) hours.
Figure 4.
Second order rate constant for nonenzymatic acetylation.
(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) kobs 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.
Figure 5.
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.
(A) Changes of modifications on KSTGGKAPR: KaSTGGKaAPR (open circle), KaSTGGKpAPR (open square), KpSTGGKaAPR (open triangle), and KpSTGGKpAPR (solid reverse triangle). (B) Changes of modifications on KQLATKAAR: KaQLATKaAAR (solid circle), KaQLATKpAAR (solid square), KpQLATKaAAR (solid triangle), and KpQLATKpAAR (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.
Figure 6.
Determination of steady-state kinetic parameters for K14 acetylation by Gcn5.
(A) kcat = 12.1±0.1 min−1 and Km = 0.5±0.02 µM are determined when [Gcn5] = 18 nM, [acetyl-CoA] = 200 μM with titrating 13 different H3 concentrations. (B) kcat = 11.6±0.2 min−1 and Km = 0.7±0.05 µM are determined when [Gcn5] = 18 nM, [H3] = 10 μM with titrating different acetyl-CoA concentrations.
Table 2.
Steady-state parameters of acetyl-CoA for Gcn5-mediated acetylation (mean ± standard error) of H3 (wt) and H3K14ac.
Table 3.
Steady-state parameters of H3 (wt) and H3K14ac for Gcn5-mediated acetylation (mean ± standard error).
Figure 7.
Determination of steady-state kinetic parameters of Gcn5-mediated acetylation of H3K14ac for individual and total lysine residues (i.e. K9, K18, and K23).
The left panels (A)-(D) show the data when titrating H3K14ac; and the right panels (E)-(H) show the data when titrating acetyl-CoA. The apparent kinetic parameters are listed in Table 2. When fitting data from (A) to (D), the Hill coefficient (nH) is approximately equal to 2, suggesting H3K14ac could form a dimer to be further acetylated.