Identification and characterization of the sodium-binding site of activated protein C
Identification and characterization of the sodium-binding site of activated protein C
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DOI：
10.1074/jbc.274.8.4970
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发表时间：
1999-02-19
影响因子：
4.8
通讯作者：
Rezaie, AR
中科院分区：
文献类型：
--
作者：
He, XH;Rezaie, AR
活化蛋白C（APC）需要Ca 2+和Na+才能发挥最佳催化功能。与Ca 2+结合位点相反，APC的Na+结合位点尚未被鉴定。基于最近对凝血酶的研究，预测221-225环是APC中潜在的Na+结合位点。该环的序列在胰蛋白酶中不保守。我们设计了一种无Gla结构域形式的蛋白C（GDPC），其中221-225环被胰蛋白酶的相应环取代。我们发现激活的GDPC（aGDPC）需要Na+（或其他碱金属阳离子）的酰胺分解活性，解离常数（K-d（app））= 44.1 +/-8.6 mM。然而，在Ca 2+存在下，aGDPC对Na+的需求被消除，Na+刺激切割速率5-6倍，K-d（app）= 2.3 +/-0.3mm。这两种阳离子都是aGDPC有效灭活因子Va所必需的。在Ca ~（2+）存在下，突变体的催化功能不依赖于Na ~+。与aGDPC不同，突变体不区分一价阳离子。我们的结论是，221-225环是一个Na+结合位点在APC和变构连接，Na+和Ca 2+结合环之间调节这种抗凝酶的结构和功能。
Activated protein C (APC) requires both Ca2+ and Na+ for its optimal catalytic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221-225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221-225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na+ (or other alkali cations) for its amidolytic activity with dissociation constant (K-d(app)) = 44.1 +/- 8.6 mM. In the presence of Ca2+, however, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated the cleavage rate 5-6-fold with K-d(app) = 2.3 +/- 0.3 mm. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221-225 loop is a Na+-binding site in APC and that an allosteric link, between the Na+ and Ca2+ binding loops modulates the structure and function of this anticoagulant enzyme.