Chen Lin, Rezaie Alireza R
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
J Biol Chem. 2004 Apr 23;279(17):17869-74. doi: 10.1074/jbc.M314285200. Epub 2004 Feb 9.
An activator complex from the venom of Oxyuranus scutellatus scutellatus (taipan venom) is known to rapidly activate prothrombin to thrombin. To determine whether, similar to prothrombinase, taipan venom utilizes proexosite-1 on prothrombin for a productive complex assembly, the activation of proexosite-1 mutants of prethrombin-1 by the partially purified venom was studied. It was discovered that basic residues of this site (Arg(35), Lys(36), Arg(67), Lys(70), Arg(73), Arg(75), and Arg(77)) are also crucial for recognition and rapid activation of the substrate by taipan venom. This was evidenced by the observation that the K(m) and k(cat) values for the activation of the charge reversal mutants of prethrombin-1 (in particular K36E, R67E, and K70E) were markedly impaired. Competitive kinetic studies with the Tyr(63)-sulfated hirudin(54-65) peptide revealed that although the peptide inhibits the activation of the wild type zymogen by taipan venom with a K(D) of approximately 2 microm, it is ineffective in inhibiting the activation of mutant zymogens (K(D) > 4-30 microm). Interestingly, an approximately 50-kDa activator, isolated from the taipan venom complex, catalyzed the activation of prothrombin in a factor Va-dependent manner and exhibited identical activation kinetics toward the substrate in the presence of the hirudin peptide. These results suggest that, similar to prothrombinase, proexosite-1 is a cofactor-dependent recognition site for taipan venom.
已知来自东部拟眼镜蛇（太攀蛇毒液）毒液的一种激活复合物能迅速将凝血酶原激活为凝血酶。为了确定太攀蛇毒液是否与凝血酶原酶类似，利用凝血酶原上的前外显子-1进行有效的复合物组装，研究了部分纯化的毒液对凝血酶原-1前体的前外显子-1突变体的激活作用。研究发现，该位点的碱性残基（Arg(35)、Lys(36)、Arg(67)、Lys(70)、Arg(73)、Arg(75)和Arg(77)）对于太攀蛇毒液识别和快速激活底物也至关重要。这一点通过观察凝血酶原-1电荷反转突变体（特别是K36E、R67E和K70E）激活的K(m)和k(cat)值明显受损得到证明。用Tyr(63)-硫酸化水蛭素(54-65)肽进行的竞争性动力学研究表明，尽管该肽以约2微摩尔的K(D)抑制太攀蛇毒液对野生型酶原的激活，但它对抑制突变体酶原的激活无效（K(D)>4-30微摩尔）。有趣的是，从太攀蛇毒液复合物中分离出一种约50 kDa的激活剂，它以因子Va依赖的方式催化凝血酶原的激活，并且在水蛭素肽存在的情况下对底物表现出相同的激活动力学。这些结果表明，与凝血酶原酶类似，前外显子-1是太攀蛇毒液的一个辅因子依赖性识别位点。
