Garza-Ramos G, Cabrera N, Saavedra-Lira E, Tuena de Gómez-Puyou M, Ostoa-Saloma P, Pérez-Montfort R, Gómez-Puyou A
Departamento de Bioquímica Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, DF.
Eur J Biochem. 1998 May 1;253(3):684-91. doi: 10.1046/j.1432-1327.1998.2530684.x.
The amino acid sequence of triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68%. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei triosephosphate isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi triosephosphate isomerase was more than 100-fold more sensitive. The sensitivity of wild type triosephosphate isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana triosephosphate isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accessibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi triosephosphate isomerase which makes its dimer interface more susceptible for perturbation.
布氏锥虫、克氏锥虫和墨西哥利什曼原虫的磷酸丙糖异构酶的氨基酸序列一致性为68%。使用布氏锥虫酶的编号系统，在它们的比对序列中，克氏锥虫和利什曼原虫的酶在第14、40、117和126位有半胱氨酸残基。布氏锥虫磷酸丙糖异构酶在第14、40和126位有半胱氨酸残基，在第117位有缬氨酸残基。二硫代硝基苯甲酸和甲基甲磺酸酯抑制这三种酶，但克氏锥虫磷酸丙糖异构酶的敏感性要高100倍以上。克氏锥虫和布氏锥虫野生型磷酸丙糖异构酶对试剂的敏感性分别与Cys117Val和Val117Cys突变酶相同。在第40和126位有半胱氨酸残基但在第14位没有半胱氨酸残基的磷酸丙糖异构酶对甲基甲磺酸酯不敏感。因此，巯基试剂作用于Cys14。在化学计量浓度下，这些试剂由于结构改变而抑制这三种酶，这种结构改变通过8-苯胺基-1-萘磺酸与先前埋藏的疏水区域的结合来衡量。然而，克氏锥虫、布氏锥虫和墨西哥利什曼原虫磷酸丙糖异构酶达到最大变化一半的时间分别为10分钟、15小时和超过30小时。pH对巯基试剂作用以及分子模拟的影响表明Cys14的溶剂可及性没有差异。由于Cys14构成二聚体界面的一部分，数据表明，在这三种酶中，不同大小的障碍阻碍了巯基试剂与Cys14之间的相互作用。克氏锥虫磷酸丙糖异构酶中的障碍较低，这使得其二聚体界面更容易受到扰动。
