Vitorino-Cardoso Ana Flávia, Pereira Ramos Oscar Henrique, Homsi-Brandeburgo Maria Inês, Selistre-de-Araujo Heloisa Sobreiro
Laboratório de Química de Proteínas e Produtos Naturais, Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia-MG, Brazil.
Comp Biochem Physiol B Biochem Mol Biol. 2006 Jul;144(3):334-42. doi: 10.1016/j.cbpb.2006.03.010. Epub 2006 Apr 3.
The cDNA encoding BthaTL, a serine peptidase from the venom of the snake Bothrops alternatus, was cloned and sequenced. The deduced primary structure shows over 62% of identity with snake venom thrombin-like enzymes (SVTLEs), molecules with high substrate specificity toward different natural substrates. Indeed, a phylogenetic reconstruction by two different methods clustered this enzyme close to other SVTLEs. These enzymes generally affect the hemostatic system in several ways, and therefore are used as tools in pharmacology and clinical diagnosis. A three-dimensional model of BthaTL was built by homology modeling using TSV-PA (Trimeresurus stejnegeri venom plasminogen activator) crystal structure as template. BthaTL model showed that the typical catalytic triad conformation of serine peptidases was preserved. The calcium coordination ligands were absent or adopt an unfavorable conformation, preventing interactions with metals. On the other hand, the Asp97-Arg174 saline bridge of TSV-PA was not found and its specificity determinant Phe193 is replaced by a Gly in BthaTL. The substitution of essential residues in the neighborhoods of the catalytic site cleft of BthaTL indicates that these two proteins do not share the same enzymatic specificity, what means that BthaTL will probably not activate plasminogen. Such observations may be helpful in the understanding of the molecular mechanism for substrate specificity of these enzymes.
编码来自交替锯鳞蝰蛇毒液的丝氨酸蛋白酶BthaTL的cDNA被克隆并测序。推导的一级结构显示与蛇毒凝血酶样酶（SVTLEs）有超过62%的同一性，这些分子对不同天然底物具有高底物特异性。实际上，通过两种不同方法进行的系统发育重建将这种酶聚类到其他SVTLEs附近。这些酶通常以多种方式影响止血系统，因此被用作药理学和临床诊断中的工具。使用竹叶青蛇毒纤溶酶原激活剂（TSV-PA）晶体结构作为模板，通过同源建模构建了BthaTL的三维模型。BthaTL模型表明丝氨酸蛋白酶的典型催化三联体构象得以保留。钙配位配体不存在或呈不利构象，阻止了与金属的相互作用。另一方面，未发现TSV-PA的Asp97-Arg174盐桥，其特异性决定簇Phe193在BthaTL中被甘氨酸取代。BthaTL催化位点裂隙附近关键残基的取代表明这两种蛋白质不具有相同的酶特异性，这意味着BthaTL可能不会激活纤溶酶原。这些观察结果可能有助于理解这些酶底物特异性的分子机制。
