Activation and Inhibition of Histone Deacetylase 8 by Monovalent Cations
Activation and Inhibition of Histone Deacetylase 8 by Monovalent Cations
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DOI：
10.1074/jbc.m109.033399
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发表时间：
2010-02-26
影响因子：
4.8
通讯作者：
Fierke, Carol A.
中科院分区：
文献类型：
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作者：
Gantt, Stephanie L.;Joseph, Caleb G.;Fierke, Carol A.
金属依赖性组蛋白去乙酰化酶（HDACs）催化乙酰赖氨酸侧链的乙酰基水解，是癌症治疗的靶点。先前在HDAC8的晶体结构中观察到两个功能未知的结合单价阳离子（MVCs）；位点1位于活性位点附近，而位点2位于距离催化金属离子100 ~ 20埃处。在这里，我们证明了一个绑定MVC激活了催化活性（K + K-1/2 = 3.4 mM），而第二个弱绑定MVC （K + K-1/2 = 26 mM）降低了催化活性11倍。较弱的结合MVC也使HDAC抑制剂亚酰苯胺羟肟酸的亲和力提高了5倍。位点1mvc由Asp-176侧链协调，Asp-176侧链也与His-142形成氢键，His-142是对催化活性很重要的两种组氨酸之一。D176A和H142A突变体分别将钾抑制的K-1/2增加了>= 40倍，表明抑制阳离子与位点1结合。此外，MVC抑制是由His-142介导的，这表明该残基被质子化以获得最大的HDAC8活性。因此，His-142既可以作为静电催化剂，也可以作为普通酸。激活的MVC结合在远端位点，并引起活性的时间依赖性增加，这表明2mvc位点稳定了酶的活性构象。钠与这两个位点的结合更弱，激活HDAC8的程度也比钾小。因此，钾很可能是体内与HDAC8结合的主要MVC。
The metal-dependent histone deacetylases (HDACs) catalyze hydrolysis of acetyl groups from acetyllysine side chains and are targets of cancer therapeutics. Two bound monovalent cations (MVCs) of unknown function have been previously observed in crystal structures of HDAC8; site 1 is near the active site, whereas site 2 is located > 20 angstrom from the catalytic metal ion. Here we demonstrate that one bound MVC activates catalytic activity (K-1/2 = 3.4 mM for K+), whereas the second, weakerbinding MVC (K-1/2 = 26 mM for K+) decreases catalytic activity by 11-fold. The weaker binding MVC also enhances the affinity of the HDAC inhibitor suberoylanilide hydroxamic acid by 5-fold. The site 1 MVC is coordinated by the side chain of Asp-176 that also forms a hydrogen bond with His-142, one of two histidines important for catalytic activity. The D176A and H142A mutants each increase the K-1/2 for potassium inhibition by >= 40-fold, demonstrating that the inhibitory cation binds to site 1. Furthermore, the MVC inhibition is mediated by His-142, suggesting that this residue is protonated for maximal HDAC8 activity. Therefore, His-142 functions either as an electrostatic catalyst or a general acid. The activating MVC binds in the distal site and causes a time-dependent increase in activity, suggesting that the site 2 MVC stabilizes an active conformation of the enzyme. Sodium binds more weakly to both sites and activates HDAC8 to a lesser extent than potassium. Therefore, it is likely that potassium is the predominant MVC bound to HDAC8 in vivo.