Kim Byungchul, Pithadia Amit S, Fierke Carol A
Chemical Biology Program, University of Michigan, Ann Arbor, Michigan, 48109-2216.
Protein Sci. 2015 Mar;24(3):354-65. doi: 10.1002/pro.2623. Epub 2015 Jan 13.
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.
组蛋白去乙酰化酶8（HDAC8）最初是根据其在体外的锌（II）依赖性HDAC8活性以及对结合到活性位点的锌（II）的鉴定，被归类为锌（II）依赖性去乙酰化酶。然而，体外测量表明，HDAC8与结合的亚铁（II）的活性高于锌（II），尽管亚铁（II）-HDAC8在有氧条件下会迅速失去活性。这些数据表明，在细胞中HDAC8可以被锌（II）或亚铁（II）激活。在这里，我们详细阐述了锌（II）和亚铁（II）与HDAC8结合的动力学、热力学和选择性。为此，我们开发了一种使用荧光素标记的辛二酰苯胺异羟肟酸（fl-SAHA）的荧光各向异性测定法。fl-SAHA与金属结合的HDAC8特异性结合，其亲和力与SAHA相当。为了测量HDAC的金属亲和力，将金属结合与fl-SAHA偶联，并根据观察到的各向异性变化进行测定。HDAC8的金属解离常数（KD）差异显著，锌（II）和亚铁（II）的KD值分别从皮摩尔到微摩尔不等。出乎意料的是，HDAC8中亚铁（II）和锌（II）的解离速率常数相当，koff约为0.0006 s-1，这表明亚铁（II）的表观缔合速率常数较慢（约3×103 M-1 s-1）。此外，结合到HDAC8的单价阳离子（K+或Na+）会使锌（II）的解离速率常数降低，K+降低≥100倍，Na+降低≥10倍，这表明体内调节金属交换的一种可能机制。HDAC8的金属亲和力与细胞中易于交换的锌（II）和亚铁（II）浓度相当，这与一种或两种金属辅因子激活HDAC8一致。
