Alleviation of intrasteric inhibition by the pathogenic activation domain mutation, D444N, in human cystathionine β-synthase
Alleviation of intrasteric inhibition by the pathogenic activation domain mutation, D444N, in human cystathionine β-synthase
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DOI：
10.1021/bi026248d
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发表时间：
2002-10-01
期刊：
影响因子：
2.9
通讯作者：
Banerjee, R
中科院分区：
文献类型：
--
作者：
Evande, R;Blom, H;Banerjee, R
人胱硫氨酸β-合成酶是一种血红素蛋白，催化丝氨酸和同型半胱氨酸在磷酸吡哆醛依赖的反应中缩合生成胱硫氨酸。这种酶的突变是遗传性高同型半胱氨酸血症的主要原因，并伴随着心血管和其他并发症。该酶被变构调节剂S-腺苷甲硫氨酸(ADOMet)激活2倍，推测ADOMet与C末端调节域结合。调控结构域对该酶具有完全的抑制作用，其缺失与酶活性增加2倍和对ADOMet的反应性丧失有关。C末端调节域的突变D444N表现出高水平的酶活性，但仍是致病的。在这项研究中，我们表征了与这种突变相关的生化惩罚，并证明它与D444N突变纯合的成纤维细胞系中稳定水平的胱硫醚β-合酶水平降低4倍有关。重组D444N酶的活性模拟了在ADOMet存在下看到的野生型酶的活性，并且在变构调节剂的超放射浓度下可以进一步被激活2倍。该突变使ADOMet的K-CAT从7.4+/-0.2微米增加到460+/-130微米，从而使该酶在生理浓度下对ADOMet没有功能反应。这些结果表明，D444N突变部分消除了C-末端结构域对空间结构的抑制作用。我们提出了一个模型，该模型考虑了人胱硫氨酸β-合酶在动力学上可区分的三种状态：“基础”(即分离的野生型酶)、“激活”(野生型酶+ADOMet或分离的D444N突变体)和超激活(D444N突变体+ADOMet或缺少C末端调节域的野生型酶)。
Human cystathionine beta-synthase is a heme protein that catalyzes the condensation of serine and homocysteine to form cystathionine in a pyridoxal phosphate-dependent reaction. Mutations in this enzyme are the leading cause of hereditary hyperhomocysteinemia with attendant cardiovascular and other complications. The enzyme is activated similar to2-fold by the allosteric regulator S-adenosylmethionine (AdoMet), which is presumed to bind to the C-terminal regulatory domain. The regulatory domain exerts ail inhibitory effect on the enzyme, and its deletion is correlated with a 2-fold increase in catalytic activity and loss of responsiveness to AdoMet. A mutation in the C-terminal regulatory domain, D444N, displays high levels of enzyme activity, yet is pathogenic. In this study, we have characterized the biochemical penalties associated with this mutation and demonstrate that it is associated with a 4-fold lower steady-state level of cystathionine beta-synthase in a fibroblast cell line that is homozygous for the D444N mutation. The activity of the recombinant D444N enzyme mimics the activity of the wild-type enzyme seen in the presence of AdoMet and can be further activated similar to2-fold in the presence of supraphysiolgical concentrations of the allosteric regulator. The mutation increases the K-cat for AdoMet from 7.4 +/- 0.2 to 460 +/- 130 muM, thus rendering the enzyme functionally unresponsive to AdoMet under physiological concentrations. These results indicate that the D444N mutation partially abrogates the intrasteric inhibition imposed by the C-terminal domain. We propose a model that takes into account the three kinetically distinguishable states that are observed with human cystathionine beta-synthase: "basal" (i.e., wild-type enzyme as isolated), "activated" (wild-type enzyme + AdoMet or the D444N mutant as isolated), and superactivated (D444N mutant + AdoMet or wild-type enzyme lacking the C-terminal regulatory domain).