KDAC8 with High Basal Velocity Is Not Activated by N-Acetylthioureas.
KDAC8 with High Basal Velocity Is Not Activated by N-Acetylthioureas.
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DOI：
10.1371/journal.pone.0146900
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Watt TJ
中科院分区：
文献类型：
--
作者：
Toro TB;Pingali S;Nguyen TP;Garrett DS;Dodson KA;Nichols KA;Haynes RA;Payton-Stewart F;Watt TJ
赖氨酸脱乙酰酶(KDAC)是一种逆转赖氨酸乙酰化翻译后修饰的酶。最近，人们合成了一系列N-乙酰基硫脲类化合物，并报道了用荧光底物来增强KDAC8的活性。为了确定活性是否具有普遍性，我们合成了三个最有效的N-乙酰基硫脲，并在多个反应条件下，利用两种酶纯化方法，测量了它们在多肽底物和荧光底物上的效果。在任何检测条件下，三种N-乙酰基硫脲均未被激活。在没有N-乙酰基硫脲的情况下，KDAC8与荧光底物的动力学进一步表征得到kcat/Km为164±17。这一催化效率与以前报道的KDAC8被N-乙酰硫脲激活时的催化效率相当或更高，这表明先前报道的激活效果可能是由于使用了含有大量失活酶的酶制剂。对活性较低的制剂和附加底物的进一步表征导致我们得出结论，N-乙酰硫脲不是KDAC8的真正激活剂，只有当酶制剂低于最大基础活性时才能提高活性。
Lysine deacetylases (KDACs) are enzymes that reverse the post-translational modification of lysine acetylation. Recently, a series of N-acetylthioureas were synthesized and reported to enhance the activity of KDAC8 with a fluorogenic substrate. To determine if the activation was general, we synthesized three of the most potent N-acetylthioureas and measured their effect with peptide substrates and the fluorogenic substrate under multiple reaction conditions and utilizing two enzyme purification approaches. No activation was observed for any of the three N-acetylthioureas under any assayed conditions. Further characterization of KDAC8 kinetics with the fluorogenic substrate yielded a kcat/KM of 164 ± 17 in the absence of any N-acetylthioureas. This catalytic efficiency is comparable to or higher than that previously reported when KDAC8 was activated by the N-acetylthioureas, suggesting that the previously reported activation effect may be due to use of an enzyme preparation that contains a large fraction of inactive enzyme. Further characterization with a less active preparation and additional substrates leads us to conclude that N-acetylthioureas are not true activators of KDAC8 and only increase activity if the enzyme preparation is below the maximal basal activity.