Chen X, Wang L, Fazlieva R, Kruger W D
Division of Population Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Hum Mutat. 2006 May;27(5):474-82. doi: 10.1002/humu.20320.
Cystathionine beta-synthase (CBS) deficiency is a recessive genetic disorder characterized by extremely elevated levels in plasma homocysteine. Patients homozygous for the I278T or R266K mutations respond clinically to pharmacologic doses of pyridoxine, the precursor of a cofactor for the enzyme, 5'-pyridoxal phosphate (PLP). Here we test the hypothesis that these mutations are pyridoxine responsive because they lower the affinity of the enzyme for PLP. We show that recombinant R266K has 30 to 100% of the specific activity of the wild-type enzyme, while I278T only has only 1 to 5% activity. Kinetic studies show that the decreased activity in both enzymes is due to reduced turnover rate and not substrate binding. Neither I278T nor R266K appear to greatly affect multimer status of the enzyme. The R266K enzyme has reduced affinity for PLP compared to the wild-type enzyme, providing a mechanism for the pyridoxine response observed in patients. Surprisingly, the I278T enzyme does not have altered affinity for PLP. To confirm that this was not an in vitro artifact, we examined pyridoxine response in mice that stably express human I278T as their sole source of CBS activity. These mice have extremely elevated plasma homocysteine levels and do not respond significantly to large doses of pyridoxine. Our findings suggest that there may be multiple mechanisms involved in response to pyridoxine.
胱硫醚β-合酶（CBS）缺乏症是一种隐性遗传疾病，其特征是血浆同型半胱氨酸水平极高。纯合子I278T或R266K突变的患者对药理剂量的吡哆醇（该酶的辅因子5'-磷酸吡哆醛（PLP）的前体）有临床反应。在这里，我们检验了这样一个假设，即这些突变对吡哆醇有反应是因为它们降低了酶对PLP的亲和力。我们发现重组R266K具有野生型酶30%至100%的比活性，而I278T仅具有1%至5%的活性。动力学研究表明，这两种酶活性的降低是由于周转率降低而非底物结合。I278T和R266K似乎都没有对酶的多聚体状态产生很大影响。与野生型酶相比，R266K酶对PLP的亲和力降低，这为患者中观察到的吡哆醇反应提供了一种机制。令人惊讶的是，I278T酶对PLP的亲和力没有改变。为了证实这不是体外假象，我们检测了稳定表达人I278T作为其唯一CBS活性来源的小鼠对吡哆醇的反应。这些小鼠的血浆同型半胱氨酸水平极高，对大剂量的吡哆醇没有明显反应。我们的研究结果表明，对吡哆醇的反应可能涉及多种机制。
