Laboratório de Química e Função de Proteínas e Peptídeos and Unidade de Experimentação Animal-RJ, CBB, UENF, Avenida Alberto Lamego 2000, Horto, Campos dos Goytacazes, RJ, CEP 28015-620, Brazil.
Insect Biochem Mol Biol. 2011 Jun;41(6):400-9. doi: 10.1016/j.ibmb.2011.02.009. Epub 2011 Mar 9.
Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 μmol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.
磷酸丙糖异构酶（TIM）是一种在糖酵解和糖异生中起作用的酶，通过催化甘油醛 3-磷酸和二羟丙酮磷酸之间的相互转化。这种酶已被用作内寄生虫药物开发的靶点。在这项工作中，我们克隆、表达、纯化并研究了来自蜱胚胎（Rhipicephalus（Boophilus）microplus）（BmTIM）的 TIM 的动力学和结构特征。重组 BmTIM 以甘油醛 3-磷酸为底物时的 Km 和 Vmax 值分别为 0.47 mM 和 6031 μmol min⁻¹ mg 蛋白⁻¹。衍射晶体的分辨率估计为 2.4 Å，整体数据表明 BmTIM 与其他报道的二聚 TIM 相似。然而，我们发现与其他 TIM 相比，BmTIM 具有最高含量的半胱氨酸残基（每个单体有九个半胱氨酸残基）。BmTIM 的单体中只有两个半胱氨酸可以形成二硫键。此外，BmTIM 对硫醇试剂二硫代硝基苯甲酸和甲基甲烷硫代磺酸的作用非常敏感，这表明每个二聚体中有五个半胱氨酸暴露，这些残基可用于开发特异性抑制剂。
