Structural and Functional Influence of the Glycine-Rich Loop G³⁰²GGGY on the Catalytic Tyrosine of Histone Deacetylase 8

Abstract

Histone deacetylase 8 (HDAC8) catalyzes the hydrolysis of acetyl-L-lysine to yield products L-lysine and acetate through a mechanism in which a nucleophilic water molecule is activated by a histidine general base and a catalytic metal ion (Zn2+ or Fe2+). Acetyl-L-lysine also requires activation by metal coordination and a hydrogen bond with catalytic tyrosine Y306, which also functions in transition state stabilization. Interestingly, Y306 is located in the conserved glycine-rich loop G302GGGY. The potential flexibility afforded by the tetraglycine segment may facilitate induced-fit conformational changes of Y306 between “in” and “out” positions, as observed in related deacetylases. To probe the catalytic importance of the glycine-rich loop in HDAC8, we rigidified this loop by preparing the G302A, G303A, G304A, and G305A mutants, and we measured their steady-state kinetics and determined their X-ray crystal structures. Substantial losses of catalytic efficiency are observed (10–500-fold based on kcat/KM), particularly for G304A HDAC8 and G305A HDAC8. These mutants also exhibit the greatest structural changes for catalytic tyrosine Y306 (1.3–1.7 Å shifts of the phenolic hydroxyl group). Molecular dynamics simulations further indicate that G304 and G305 undergo pronounced structural changes as residue 306 transitions between “in” and “out” conformations. Thus, the G304A and G305A substitutions likely compromise the position and conformational changes of Y306 required for substrate activation and transition state stabilization. The G302A and G303A substitutions have less severe catalytic consequences, and these substitutions may influence an internal channel through which product acetate is believed to exit.

Lysine Deacetylases Exhibit Distinct Changes in Activity Profiles Due to Fluorophore Conjugation of Substrates

“…For the metal-dependent KDACs tested, the presence of AMC led to a large increase in activity (Figure 2 and Table S2). These results are consistent with previously reported data for KDAC8 with another set of peptides assayed using a distinct method, 28 and the values of k cat and K M we determined are consistent with available prior data for some of the AMC-containing peptides, 3,38-40 with variation in k cat related to known effects of different reaction conditions that we have previously reported on. 26,41 KDAC6 was previously shown to exhibit greater endpoint activity with a peptide containing C-terminal acetyllysine compared to a corresponding peptide with AMC in the C-terminal position.…”

Binding of the Microbial Cyclic Tetrapeptide Trapoxin A to the Class I Histone Deacetylase HDAC8

“…18 Among the metal-dependent HDACs, HDAC8 was the first to yield a crystal structure. 19,20 Important catalytic residues in the HDAC8 active site include tandem histidines (electrostatic catalyst H142 and general base-general acid H143) 21,22 and a conformationally-flexible 23 tyrosine residue that assists the Zn 2+ ion in the polarization of the substrate carbonyl group. 24,25 …”

Chelatable trace zinc causes low, irreproducible KDAC8 activity

“…The need to overcome this trace zinc may also account for why most prior studies of KDAC8 utilized enzyme concentrations much greater than required under our conditions. 8,9,23–25,29,30,37–39 Indeed, in tris-based buffer (but not in phosphate-based buffer), the specific activity of KDAC8 was much lower at low enzyme concentrations; however, this effect could be eliminated by addition of BSA (Figures S3 and 1C). Critically, even a 500-fold excess of EDTA did not impair enzyme activity during the course of the reaction (Figure 2A), indicating that the chelator is not sufficient to strip the catalytic metal from the enzyme under reaction conditions.…”