Tethered processivity of the vitamin K-dependent carboxylase: factor IX is efficiently modified in a mechanism which distinguishes Gla's from Glu's and which accounts for comprehensive carboxylation in vivo.
Tethered processivity of the vitamin K-dependent carboxylase: factor IX is efficiently modified in a mechanism which distinguishes Gla's from Glu's and which accounts for comprehensive carboxylation in vivo.
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维生素 K 依赖性羧化酶的束缚持续合成能力：因子 IX 在一种区分 Glas 和 Glus 的机制中得到有效修饰，并解释了体内全面的羧化作用。
DOI：
10.1021/bi0107039
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Berkner,KL
中科院分区：
文献类型：
--
作者：
Stenina,O;Pudota,BN;McNally,BA;Hommema,EL;Berkner,KL
维生素K依赖性（VKD）羧化酶通过VKD蛋白的前肽结合VKD蛋白，并在Gla结构域中将Glu转化为γ-羧化Glu或Gla。活性需要多重羧化，如果羧化酶是进行性的，则可以实现多重羧化。在之前唯一一项测试这种能力的研究中，使用了间接测定法，这表明了持续合成能力;然而，效率很差，并提出了关于如何完成完全羧化的问题。为了明确地确定羧化酶是否是进行性的，以及它是否可以解释体内的全面羧化，以及阐明酶的机制，我们开发了一种直接的持续性测试。用过量的可区分的fIX变体挑战含有羧化酶和全长因子IX（fIX）的复合物的体外羧化。值得注意的是，复合物中fIX的羧化完全不受攻击蛋白的影响，并且实现了全面的羧化，最终表明羧化酶是进行性的和高效的。这些研究还表明，个别fIX/羧化酶复合物的羧化是不同步的，并暗示了需要羧化酶区分Glu与Gla的反应的驱动力。我们发现Gla结构域在羧化过程中与羧化酶紧密结合，阻断了小肽底物（EEL）的进入。该研究描述了预形成的复合物的第一次分析，并且复合物中全长天然fIX的速率与底物EEL的速率相当。因此，Gla结构域内的分子内移动以重新定位用于催化的新Glu与小底物的扩散限制定位一样快，并且Gla结构域在羧化期间不受fIX分子的其余部分的空间限制。预形成的复合物中的fIX的羧化速率比通过游离羧化酶修饰的fIX高24倍，这支持羧化酶的持续合成能力，并且表明结合和/或释放是蛋白质羧化中的限速步骤。这些数据表明系留持续合成能力的模型，其中VKD蛋白通过其前肽在整个反应中保持与羧化酶结合，而Gla结构域经历分子内运动以重新定位新的Glu用于催化，从而最终实现全面的羧化。
The vitamin K-dependent (VKD) carboxylase binds VKD proteins via their propeptide and converts Glu's to γ-carboxylated Glu's, or Gla's, in the Gla domain. Multiple carboxylation is required for activity, which could be achieved if the carboxylase is processive. In the only previous study to test for this capability, an indirect assay was used which suggested processivity; however, the efficiency was poor and raised questions regarding how full carboxylation is accomplished. To unequivocally determine if the carboxylase is processive and if it can account for comprehensive carboxylation in vivo, as well as to elucidate the enzyme mechanism, we developed a direct test for processivity. The in vitro carboxylation of a complex containing carboxylase and full-length factor IX (fIX) was challenged with an excess amount of a distinguishable fIX variant. Remarkably, carboxylation of fIX in the complex was completely unaffected by the challenge protein, and comprehensive carboxylation was achieved, showing conclusively that the carboxylase is processive and highly efficient. These studies also showed that carboxylation of individual fIX/carboxylase complexes was nonsynchronous and implicated a driving force for the reaction which requires the carboxylase to distinguish Glu's from Gla's. We found that the Gla domain is tightly associated with the carboxylase during carboxylation, blocking the access of a small peptide substrate (EEL). The studies describe the first analysis of preformed complexes, and the rate for full-length, native fIX in the complex was equivalent to that of the substrate EEL. Thus, intramolecular movement within the Gla domain to reposition new Glu's for catalysis is as rapid as diffusion-limited positioning of a small substrate, and the Gla domain is not sterically constrained by the rest of the fIX molecule during carboxylation. The rate of carboxylation of fIX in the preformed complex was 24-fold higher than for fIX modified by free carboxylase, which supports carboxylase processivity and which indicates that binding and/or release is the rate-limiting step in protein carboxylation. These data indicate a model of tethered processivity, in which the VKD proteins remain bound to the carboxylase throughout the reaction via their propeptide, while the Gla domain undergoes intramolecular movement to reposition new Glu's for catalysis to ultimately achieve comprehensive carboxylation.