Synthesis of amino acid cofactor in cysteine dioxygenase is regulated by substrate and represents a novel post-translational regulation of activity
Synthesis of amino acid cofactor in cysteine dioxygenase is regulated by substrate and represents a novel post-translational regulation of activity
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DOI：
10.1074/jbc.m800044200
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发表时间：
2008-05-02
影响因子：
4.8
通讯作者：
Stipanuk, Martha H.
中科院分区：
文献类型：
--
作者：
Dominy, John E.;Hwang, Jesse;Stipanuk, Martha H.
半胱氨酸双加氧酶（CDO）催化半胱氨酸转化为半胱氨酸亚磺酸，并且在调节哺乳动物细胞内半胱氨酸水平和提供氧化的半胱氨酸代谢物如硫酸盐和牛磺酸中是重要的。哺乳动物CDO的几个晶体结构的研究表明，有一个交联的辅因子存在于酶的活性位点。辅因子由残基半胱氨酸93的γ-硫和残基酪氨酸157的芳族侧链之间的硫醚键组成。辅因子合成的确切要求和辅因子对酶催化活性的贡献尚未完全描述。因此，在这项研究中，我们探讨了在体外和体内的辅因子生物合成所必需的因素，并研究了辅因子的形成对体外活性的影响。与其他含交联辅因子的酶一样，CDO中Cys-Tyr辅因子的形成需要过渡金属辅因子（Fe 2+）和O-2。然而，与其他酶不同的是，生物合成也严格依赖于底物的存在。辅因子的形成也明显慢于其他酶的速率，事实上，需要数百个催化周转周期才能发生。在没有Cys-Tyr辅因子的情况下，CDO具有明显的催化活性，表明辅因子不是催化所必需的。然而，在生理相关的半胱氨酸浓度，辅因子的形成增加了CDO催化效率的10倍。总体而言，环境半胱氨酸水平对CDO中Cys-Tyr辅因子形成的调节代表了具有重要生理后果的底物介导的前馈激活酶活性的不寻常形式。
Cysteine dioxygenase (CDO) catalyzes the conversion of cysteine to cysteinesulfinic acid and is important in the regulation of intracellular cysteine levels in mammals and in the provision of oxidized cysteine metabolites such as sulfate and taurine. Several crystal structure studies of mammalian CDO have shown that there is a cross-linked cofactor present in the active site of the enzyme. The cofactor consists of a thioether bond between the gamma-sulfur of residue cysteine 93 and the aromatic side chain of residue tyrosine 157. The exact requirements for cofactor synthesis and the contribution of the cofactor to the catalytic activity of the enzyme have yet to be fully described. In this study, therefore, we explored the factors necessary for cofactor biogenesis in vitro and in vivo and examined what effect cofactor formation had on activity in vitro. Like other cross-linked cofactor-containing enzymes, formation of the Cys-Tyr cofactor in CDO required a transition metal cofactor (Fe2+) and O-2. Unlike other enzymes, however, biogenesis was also strictly dependent upon the presence of substrate. Cofactor formation was also appreciably slower than the rates reported for other enzymes and, indeed, took hundreds of catalytic turnover cycles to occur. In the absence of the Cys-Tyr cofactor, CDO possessed appreciable catalytic activity, suggesting that the cofactor was not essential for catalysis. Nevertheless, at physiologically relevant cysteine concentrations, cofactor formation increased CDO catalytic efficiency by similar to 10-fold. Overall, the regulation of Cys-Tyr cofactor formation in CDO by ambient cysteine levels represents an unusual form of substrate-mediated feed-forward activation of enzyme activity with important physiological consequences.