title: Kinetics and thermodynamics of metal-binding to histone deacetylase 8.
abstract: Histone deacetylase 8 (HDAC8) was originally classified as a Zn(II)-dependent deacetylase on the basis of Zn(II)-dependent HDAC8 activity in vitro and illumination of a Zn(II) bound to the active site. However, in vitro measurements demonstrated that HDAC8 has higher activity with a bound Fe(II) than Zn(II), although Fe(II)-HDAC8 rapidly loses activity under aerobic conditions. These data suggest that in the cell HDAC8 could be activated by either Zn(II) or Fe(II). Here we detail the kinetics, thermodynamics, and selectivity of Zn(II) and Fe(II) binding to HDAC8. To this end, we have developed a fluorescence anisotropy assay using fluorescein-labeled suberoylanilide hydroxamic acid (fl-SAHA). fl-SAHA binds specifically to metal-bound HDAC8 with affinities comparable to SAHA. To measure the metal affinity of HDAC, metal binding was coupled to fl-SAHA and assayed from the observed change in anisotropy. The metal KD values for HDAC8 are significantly different, ranging from picomolar to micromolar for Zn(II) and Fe(II), respectively. Unexpectedly, the Fe(II) and Zn(II) dissociation rate constants from HDAC8 are comparable, koff ∼0.0006 s(-1), suggesting that the apparent association rate constant for Fe(II) is slow (∼3 × 10(3) M(-1) s(-1)). Furthermore, monovalent cations (K(+) or Na(+)) that bind to HDAC8 decrease the dissociation rate constant of Zn(II) by ≥100-fold for K(+) and ≥10-fold for Na(+), suggesting a possible mechanism for regulating metal exchange in vivo. The HDAC8 metal affinities are comparable to the readily exchangeable Zn(II) and Fe(II) concentrations in cells, consistent with either or both metal cofactors activating HDAC8.
authors.affiliation: Chemical Biology Program, University of Michigan, Ann Arbor, Michigan, 48109-2216.
authors.forename: Byungchul
authors.initials: B
authors.lastname: Kim
authors.forename: Amit S
authors.initials: AS
authors.lastname: Pithadia
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D006655:Histone Deacetylases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D012097:Repressor Proteins; D007501:Iron; C409470:HDAC8 protein, human; D006655:Histone Deacetylases; D015032:Zinc
keywords: fl-SAHA; histone deacetylase 8; metal-binding mechanism; monovalent cations