Kinetics and Comparative Reactivity of Human Class I and Class IIb Histone Deacetylases

Abstract

Histone deacetylase (HDAC) enzymes modulate gene expression through the deacetylation of acetylated lysine residues on histone proteins. They operate in biological systems as part of multiprotein corepressor complexes. To understand the reactivity of isolated HDACs and the contribution of cofactor binding to reactivity, the reaction kinetics of isolated, recombinant human HDACs 1, 2, 3, 6, 8, and 10 were measured using a novel, continuous protease-coupled enzyme assay. Values of k(cat) and k(cat)/K(m) and the pH dependence of these values were determined for the reactions of each isozyme with acetyl-Gly-Ala-(N(epsilon)-acetyl-Lys)-AMC. Values of k(cat) spanned the range of 0.006-2.8 s(-1), and k(cat)/K(m) values ranged from 60 to 110000 M(-1) s(-1). The pH profiles for both k(cat) and k(cat)/K(m) were bell-shaped for all of the HDAC isozymes, with pH optima at approximately pH 8. Values of K(i) for the inhibitor trichostatin A were determined for each isozyme. The inhibition constants were generally similar for all HDAC isozymes, except that the value for HDAC8 was significantly higher than that for the other isozymes. The reaction of HDAC8 with an alternative substrate was performed to assess the steric requirements of the HDAC8 active site, and the effect of phosphorylation on HDAC1 activity was examined. The results are discussed in terms of the biological roles of the HDAC enzymes and the proposed reaction mechanism of acetyllysine hydrolysis by these enzymes.

Mechanism of N-Acylthiourea-mediated Activation of Human Histone Deacetylase 8 (HDAC8) at Molecular and Cellular Levels

“…TM-2-51 and coumarin-SAHA were synthesized in our laboratory using previously published protocols (22,29). SAHA, Fluor-de-Lys (KI-104 (Fig.…”

“…Steady-state Kinetics of the HDAC8-catalyzed Reaction-The steady-state kinetic studies for the HDAC8-catalyzed reaction were performed using Fluor-de-Lys as the fluorogenic substrate via a trypsin-coupled assay as described previously (29). The initial rate of the HDAC8-catalyzed reaction was determined as a function of the substrate concentration in the absence and the presence of varying concentrations of the activator.…”

A novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces apoptosis in T-cell lymphomas

“…26 For inhibitor characterization, measurements were performed in a reaction volume of 100 ml À1 using 96-well assay plates in a fluorescence plate reader. For each isozyme, the HDAC protein in reaction buffer (50 mM HEPES, 100 mM KCl, 0.001% Tween-20, 5% dimethyl sulfoxide , pH 7.4, supplemented with bovine serum albumin at concentrations of 0-0.05%) was mixed with inhibitor at various concentrations and allowed to incubate for 15 min.…”

CRA-024781: a novel synthetic inhibitor of histone deacetylase enzymes with antitumor activity in vitro and in vivo

“…HDAC Activity HDAC activity was measured using a continuous trypsin-coupled assay that has been previously described in detail (11). For inhibitor characterization, measurements were done in a reaction volume of 100 AL using 96-well assay plates.…”