Histone Deacetylase 8: Characterization of Physiological Divalent Metal Catalysis

Abstract

Histone deacetylases (HDACs) are responsible for the removal of acetyl groups from histones, resulting in gene silencing. Overexpression of HDACs is associated with cancer, and their inhibitors are of particular interest as chemotherapeutics. However, HDACs remain a target of mechanistic debate. HDAC class 8 is the most studied HDAC, and of particular importance due to its human oncological relevance. HDAC8 has traditionally been considered to be a Zn-dependent enzyme. However, recent experimental assays have challenged this assumption and shown that HDAC8 is catalytically active with a variety of different metals, and that it may be a Fedependent enzyme in vivo. We studied two opposing mechanisms utilizing a series of divalent metal ions in physiological abundance (Zn ). Extensive sampling of the entire protein with different bound metals was done with the mixed quantum-classical QM/DMD method. Density functional theory (DFT) on an unusually large cluster model was used to describe the active site and reaction mechanism. We have found that the reaction profile of HDAC8 is similar among all metals tested, and follows one of the previously published mechanisms, but the rate-determining step is different from the one previously claimed. We further provide a scheme for estimating the metal binding affinities to the protein. We use the quantum theory of atoms in molecules (QTAIM) to understand the different binding affinities for each metal in HDAC8 as well as the ability of each metal to bind and properly orient the substrate for deacetylation. The combination of this data with the catalytic rate constants is required to reproduce the experimentally observed trend in metal-depending performance. We predict Co 2+ and Zn 2+ to be the most active metals in HDAC8, followed by Fe 2+ , and Mn 2+ and Mg 2+ to be the least active. ■ INTRODUCTIONThe acetylation of lysine residues is an important reversible post-translational modification that modulates protein function, affecting a variety of cellular processes. 1−5 Proteomic surveys 6−8 have identified acetyl-lysine residues in diverse groups of proteins, including transcription factors, 9,10 cell signaling proteins, 11 metabolic enzymes (most prominently acetyl-CoA synthase 12−14 ), structural proteins in the cytoskeleton, 15,16 and HIV viral proteins. 17,18 One of the first discovered examples of lysine acetylation was that occurring in histones, 19,20 the predominant protein components of chromatin. Acetylation of histones has been linked to gene regulation: the addition of an acetyl moiety to histone lysine residues gives rise to an open chromatin structure that facilitates DNA transcription, while the removal of acetyl from histone acetyl-lysine residues is associated with a closed chromatin structure, transcriptional repression, and gene silencing. 21 The enzymes responsible for the addition and removal of acetyl groups are known as histone acetyltransferases (HATs) and histone deacetylases (HDACs) 22−26 for historical reasons, although it is now recog...

Quantified electrostatic preorganization in enzymes using the geometry of the electron charge density

“…S1 in ESI † ). 16 Details on QM/DMD can be found elsewhere. 17 The largest active site models were determined by including any residue with at least one atom within 7 Å of the Zn 2+ ion.…”

“…1 to be the most viable. 16 Through this mechanism, H143 acts first as a base, abstracting a proton from the water molecule, and second as an acid, transferring the proton to the substrate.…”

“…Furthermore, the amount of charge density at the bond critical point (CP), a saddle point in ρ , between the zinc ion and a nitrogen atom in H180 (NHis) was found to be linearly correlated to metal binding affinity for HDAC8. 16 In this study, we thus focus on the charge density around these five atoms, which are highlighted in Fig. 2 .…”

Predictive methods for computational metalloenzyme redesign – a test case with carboxypeptidase A

“…Dr(r) in the reactant states of related enzymes, for example designed computationally to catalyze the same reaction, serves as a probe of relative catalytic activity prior to the mechanistic study, as is shown here, and also was shown in a recent study of the metal-specificity in histone deacetylase. 96 This method is motivated by the Hammond postulate, which states that reaction barriers will be lower when the reactant state is ''similar'' to the transition state. The extensions to QTAIM used here provide a method for quantifying the similarity of reactant and transition states, through the amount of charge density at critical points that play an important role in the reaction coordinate. )…”

Revealing electronic features governing hydrolysis of cephalosporins in the active site of the L1 metallo-β-lactamase

“…Molecular modeling in combination with electron-density analysis is already used for organic reaction studies. Equilibrium geometry congurations and corresponding calculated electron densities of reagents, intermediates, transition states and products of chemical reactions are analyzed by various approaches as QTAIM (Quantum Theory of Atoms in Molecules) [29][30][31][32][33][34][35][36][37][38][39][40][41] or conceptual DFT (Density Functional Theory) 23,36,[42][43][44] as well as diverse bonding descriptors. [33][34][35][36][37]45,46 Current studies of enzymatic reactions [47][48][49] also demonstrate an enhanced interest in the application of such combined methods in biochemistry.…”