Kini R Manjunatha
Protein Science Laboratory, Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore.
Toxicon. 2005 Jun 15;45(8):1147-61. doi: 10.1016/j.toxicon.2005.02.018. Epub 2005 Apr 13.
Phospholipase A(2) (PLA(2)) enzymes from snake venom are toxic and induce a wide spectrum of pharmacological effects, despite similarity in primary, secondary and tertiary structures and common catalytic properties. Thus, the structure-function relationships and the mechanism of this group of small proteins are subtle, complex and intriguing challenges. This review, taking the PLA(2) enzymes from spitting cobra (Naja nigricollis) venom as examples, describes the mechanism of anticoagulant effects. The strongly anticoagulant CM-IV inhibits both the extrinsic tenase and prothrombinase complexes, whereas the weakly anticoagulant PLA(2) enzymes (CM-I and CM-II) inhibit only the extrinsic tenase complex. CM-IV binds to factor Xa and interferes in its interaction with factor Va and the formation of prothrombinase complex. In contrast, CM-I and CM-II do not affect the formation of prothrombinase complex. In addition, CM-IV inhibits the extrinsic tenase complex by a combination of enzymatic and nonenzymatic mechanisms, while CM-I and CM-II inhibit by only enzymatic mechanism. These functional differences explain the disparity in the anticoagulant potency of N. nigricollis PLA(2) enzymes. Similarly, human secretory enzyme binds to factor Xa and inhibits the prothrombinase complex. We predicted the anticoagulant region of PLA(2) enzymes using a systematic and direct comparison of amino acid sequences. This region between 54 and 77 residues is basic in the strongly anticoagulant PLA(2) enzymes and neutral or negatively charged in weakly and non-anticoagulant enzymes. The prediction is validated independently by us and others using both site directed mutagenesis and synthetic peptides. Thus, strongly anticoagulant CM-IV binds to factor Xa (its target protein) through the specific anticoagulant site on its surface. In contrast, weakly anticoagulant enzymes, which lack the anticoagulant region fail to bind specifically to the target protein, factor Xa in the coagulation cascade. Thus, these studies strongly support the target model which suggests that protein-protein interaction rather than protein-phospholipid interaction determines the pharmacological specificity of PLA(2) enzymes.
尽管蛇毒中的磷脂酶A2（PLA2）酶在一级、二级和三级结构以及常见催化特性上具有相似性，但它们具有毒性并能引发广泛的药理作用。因此，这组小蛋白质的结构-功能关系以及作用机制是微妙、复杂且引人入胜的挑战。本综述以喷毒眼镜蛇（黑颈眼镜蛇）毒液中的PLA2酶为例，描述了其抗凝作用机制。强抗凝性的CM-IV可抑制外源性凝血酶原酶复合物和凝血酶原酶复合物，而弱抗凝性的PLA2酶（CM-I和CM-II）仅抑制外源性凝血酶原酶复合物。CM-IV与因子Xa结合并干扰其与因子Va的相互作用以及凝血酶原酶复合物的形成。相比之下，CM-I和CM-II不影响凝血酶原酶复合物的形成。此外，CM-IV通过酶促和非酶促机制的组合抑制外源性凝血酶原酶复合物，而CM-I和CM-II仅通过酶促机制抑制。这些功能差异解释了黑颈眼镜蛇PLA2酶抗凝效力的差异。同样，人分泌型酶与因子Xa结合并抑制凝血酶原酶复合物。我们通过对氨基酸序列进行系统直接比较，预测了PLA2酶的抗凝区域。在强抗凝性的PLA2酶中，该54至77个残基之间的区域呈碱性，而在弱抗凝性和非抗凝性酶中呈中性或带负电荷。我们和其他人通过定点诱变和合成肽独立验证了该预测。因此，强抗凝性的CM-IV通过其表面的特定抗凝位点与因子Xa（其靶蛋白）结合。相比之下，缺乏抗凝区域的弱抗凝性酶无法在凝血级联反应中与靶蛋白因子Xa特异性结合。因此，这些研究有力地支持了靶标模型，该模型表明蛋白质-蛋白质相互作用而非蛋白质-磷脂相互作用决定了PLA2酶的药理特异性。
