Catalytic mechanism of a MYST family histone acetyltransferase

Abstract

Distinct catalytic mechanisms have been proposed for the Gcn5 and MYST histone acetyltransferase (HAT) families. Gcn5-like HATs utilize an ordered sequential mechanism involving direct nucleophilic attack of the N-epsilon-lysine on the enzyme-bound acetyl-CoA. Recently, MYST enzymes were reported to employ a ping-pong route of catalysis via an acetyl-cysteine intermediate. Here, using the prototypical MYST family member Esa1, and its physiological complex (piccolo NuA4), steady-state kinetic analyses revealed a kinetic mechanism that requires the formation of a ternary complex prior to catalysis, where acetyl-CoA binds first and CoA is the last product released. In the absence of histone acceptor, slow rates of enzyme auto-acetylation (7 x 10(-4) s(-1), or approximately 2500-fold slower than histone acetylation; kcat = 1.6 s(-1)) and of CoA formation (0.0021 s(-1)) were inconsistent with a kinetically competent acetyl-enzyme intermediate. Previously, Cys-304 of Esa1 was the proposed nucleophile that forms an acetyl-cysteine intermediate. Here, mutation of this cysteine (C304A) in Esa1 or within the piccolo NuA4 complex yielded an enzyme that was catalytically indistinguishable from the wild type. Similarly, a pH rate (kcat) analysis of the wild type and C304A revealed an ionization (pKa = 7.6-7.8) that must be unprotonated. Mutation of a conserved active-site glutamate (E338Q) reduced kcat approximately 200-fold at pH 7.5; however, at higher pH, E338Q exhibited nearly wild-type activity. These data are consistent with Glu-338 (general base) activating the N-epsilon-lysine by deprotonation. Together, the results suggest that MYST family HATs utilize a direct-attack mechanism within an Esa1 x acetyl-CoA x histone ternary complex.

Figure 1. picNuA4 requires a ternary complex for histone acetylation

(A) Double reciprocal plot from bi-substrate experiment. Acetyltransferase reaction conditions were 50 mM Tris, pH 7.5, 1 mM DTT, 0.1 μM picNuA4 with coupled assay conditions as described in Berndsen and Denu (20). Acetyl-CoA concentrations are 0.25 μM (circles), 0.5 μM (boxes), 1 μM (downward triangles), and 10 μM (right triangles) with H4₁₋₂₀ varied from 50 to 1800 μM. Data were fitted to equation 1 for a sequential mechanism in Kinetasyst and depicted in Kaleidagraph. Experiments were repeated in triplicate with representative experiment shown. (B)—Slope replot from bisubstrate experiment using propionyl-CoA and H4₁₋₂₀. DTNB assay conditions were 50 mM Tris, pH 7.5 with 1 mM EDTA with 0.1 μM picNuA4. Propionyl-CoA was varied from 10−104 μM with H4₁₋₂₀ varied from 36−550 μM. Curves were fitted to the Michaelis-Menten equation (v=V_m*[S]/(K_m + [S])) in Kaleidagraph to determine k_cat and k_cat/K_m. The 1/k_cat/K_m value with propionyl-CoA was then plotted versus 1/[H4₁₋₂₀]. Experiments were repeated in duplicate with average data ± standard deviation shown. (C)—Double reciprocal plot showing CoA inhibition when acetyl-CoA is varied at constant peptide concentration. Reaction conditions were 50 mM Tris, pH 7.5, 1 mM DTT, 0.03 μM picNuA4, and 500 μM H4₁₋₂₀. Acetyl-CoA was varied from 1 to 10 μM at 0 (circles), 2.5 (right triangles), 5 (downward triangles), and 18.2 (boxes) μM CoA. Data were fitted to equation 2 for competitive inhibition and depicted using Kaleidagraph. The K_i for CoA was determined to be 1.8 ± 0.8 μM. Experiments were repeated in duplicate with representative data shown.

Figure 2. pH profile of picNuA4 and mutants

Assays of picNuA4 activity were performed under saturating conditions for both substrates (75 μM acetyl-CoA, 1 mM H4₁₋₂₀). Reactions were performed in either 50 mM Tris, 50 mM bis-Tris, 100 mM sodium acetate or 50 mM Tris, 50 mM ethanolamine, 100 mM ACES buffer from pH 5.5 to 10.5. Data for the Wild-type enzyme are denoted with boxes, C304A with circles, and E338Q with triangles. Data were fitted to equation 3 and depicted using Kaleidagraph. The pK_a value determined for the wild-type complex was determined to be 7.8 ± 0.1, for C304A 7.6 ± 0.2, and for E338Q 9.2 ± 0.1. Experiments were repeated in duplicate or triplicate with representative curves for each enzyme shown.

Figure 3. Direct attack mechanism for acetyl-transfer by Esa1

After binding acetyl-CoA and peptide substrate to form a ternary complex, Glutamate 338 of Esa1 deprotonates the ε-amine of lysine in the substrate. Lysine attacks the carbonyl carbon of the acetyl moiety of acetyl-CoA forming a tetrahedral intermediate, which then collapses to form CoA-SH and acetylated product.