Kinetics and comparative reactivity of human class I and class IIb histone deacetylases
Kinetics and comparative reactivity of human class I and class IIb histone deacetylases
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DOI：
10.1021/bi0494471
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发表时间：
2004-08-31
期刊：
影响因子：
2.9
通讯作者：
Wong, L
中科院分区：
文献类型：
--
作者：
Schultz, BE;Misialek, S;Wong, L
组蛋白脱乙酰酶(HDAC)酶通过组蛋白上乙酰化赖氨酸残基的脱乙酰化来调节基因的表达。它们在生物系统中作为多蛋白辅阻遏子复合体的一部分工作。为了了解分离的HDAC的反应性以及辅因子结合对反应性的贡献，用一种新的、连续的蛋白酶偶联酶试验测定了分离的、重组的人HDAC 1、2、3、6、8和10的反应动力学。测定了各同工酶与乙酰甘氨酸-丙氨酸-(N-epsilon-乙酰-赖氨酸)-AMC反应的k(CAT)和k(CAT)/K-m值及其与pH的依赖关系。K(CAT)的取值范围为0.006-2.8M-1 S(-1)，k(CAT)/K-m，取值范围为60-110000 M-1 S(-1)。对所有的HDAC同工酶，k(CAT)和k(CAT)/K-m的pH曲线均为钟形，最适pH约为8。测定了各同工酶抑制剂曲古菌素A的KI值。除HDAC8的抑制常数显著高于其他同工酶外，其余各同工酶的抑制常数基本相似。通过HDAC8与另一种底物的反应来评估HDAC8活性中心的空间需求，并检测了磷酸化对HDAC1活性的影响。根据HDAC酶的生物学作用和提出的这些酶对乙酰赖氨酸的反应机理，对结果进行了讨论。
Histone deacetylase (HDAC) enzymes modulate gene expression through the deacetylation of acetylated lysine residues on histone proteins. They operate in biological systems as part of multiprotein corepressor complexes. To understand the reactivity of isolated HDACs and the contribution of cofactor binding to reactivity, the reaction kinetics of isolated, recombinant human HDACs 1, 2, 3, 6, 8, and 10 were measured using a novel, continuous protease-coupled enzyme assay. Values of k(cat) and k(cat)/K-m and the pH dependence of these values were determined for the reactions of each isozyme with acetyl-Gly-Ala-(N-epsilon-acetyl-Lys)-AMC. Values of k(cat) spanned the range of 0.006-2.8 s(-1), and k(cat)/K-m, values ranged from 60 to 110000 M-1 s(-1). The pH profiles for both k(cat) and k(cat)/K-m were bell-shaped for all of the HDAC isozymes, with pH optima at approximately pH 8. Values of K-i for the inhibitor trichostatin A were determined for each isozyme. The inhibition constants were generally similar for all HDAC isozymes, except that the value for HDAC8 was significantly higher than that for the other isozymes. The reaction of HDAC8 with an alternative substrate was performed to assess the steric requirements of the HDAC8 active site, and the effect of phosphorylation on HDAC1 activity was examined. The results are discussed in terms of the biological roles of the HDAC enzymes and the proposed reaction mechanism of acetyllysine hydrolysis by these enzymes.