a commercial acetyllysine-fluorophore substrate. Deacetylation of the acetyllysine-fluorophore substrate is followed by cleavage of the lysine-fluorophore amide bond by a protease developer, resulting in a fluorescence shift
polyamines are essential aliphatic polycations that bind to nucleic acids and accordingly are involved in a variety of cellular processes. Polyamine function can be regulated by acetylation and deacetylation, just as histone function can be regulated by lysine acetylation and deacetylation
the enzyme adopts the characteristic arginase-deacetylase fold and employ a Zn2+-activated water molecule for catalysis. The active sites of HDAC10 (histone 10 deacetylase, EC 18.104.22.168) and APAH are sterically constricted to enforce specificity for long, slender polyamine substrates and exclude bulky peptides and proteins containing acetyl-L-lysine. The quaternary structure, i.e., dimer assembly, provides the steric constriction that directs the polyamine substrate specificity of APAH. Structure and catalytic mechanism of polyamine deacetylases, comparison of HDAC and APAH, overview
nucleophilic attack of Zn2+-bound water at the amide carbonyl group polarized by Zn2+ and the catalytic tyrosine is facilitated by a general base. The Zn2+ ion, tyrosine, and tandem histidine residues contribute to transition state stabilization in each deacetylase. Collapse of the tetrahedral intermediate requires a proton donor, and the second histidine of the tandem pair must serve as the general acid due to its proximity to the leaving amino group. Structure-function analysis of substrate specificity, overview
proposed mechanism of APAH, overview. The substrate binding mode is based on the complex of N8-acetylspermidine with the inactive mutant APAH H159A (PDB entry 3Q9C), and the binding of the tetrahedral intermediate is mimicked by the binding of trifluoromethylketone inhibitor 1
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purified enzyme in complex with 5 different inhibitors, sitting drop vapor diffusion method, mixing of 500 nl of 5-10 mg/ml of protein in 20 mM Tris, pH 8.0, 50 mM NaCl, 0.01% sodium azide, 0.5-2 mM ZnCl2, and 2.5 mM inhibitor, with 500 nl of precipitant solution containing 0.2 M LiNO3, or KNO3, or LiCL, and 20% w/v PEG 3350, and equilibration against 0.1 ml of precipitant solution reservoir, 2 days, 21°C, X-ray diffraction structure determination and analysis at 1.13-1.42 A resolution