Ramírez-Silva Leticia, Riveros-Rosas Héctor, Hernández-Alcántara Gloria, García-Trejo José J, Vega-Segura Alicia, González-Andrade Martin, Díaz-Salazar A Jessica, Salcedo-Barrientos Guillermo
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico.
Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico.
Int J Mol Sci. 2025 Aug 29;26(17):8410. doi: 10.3390/ijms26178410.
Phylogenetic studies of the pyruvate kinase family reveal two clusters: the K-dependent and -independent enzymes. pyruvate kinase belongs to the latter but possesses the conserved signature of those K-dependent. Recently, we found two distinct ways for these groups to catalyze. It is interesting to elucidate how the enzyme achieves its active conformation. A structural model of this enzyme revealed the presence of an extra C-terminal sequence (ECTS). To understand its role, an enzyme lacking this sequence from was constructed. We then compared the kinetic parameters, far-UV CD spectra, thermal stability, molecular dynamics simulations, and oligomeric states of both the wild-type and truncated enzymes. We found that the truncated enzyme is aggregated and almost inactive, with residual 20% of the total interactions, and it exhibits a soluble fraction of smaller oligomeric states than the wild-type enzyme. These findings suggest that ECTS plays a crucial role in maintaining its active tetrameric state. This sequence is the first reported in an archaeal pyruvate kinase and is also found in other archaea and bacteria. Phylogenetic analysis of ECTS in pyruvate kinases exhibits a sparse distribution that might be explained if ECTS represents an ancient domain prone to loss.
依赖K⁺和不依赖K⁺的酶。丙酮酸激酶属于后者，但具有那些依赖K⁺的酶的保守特征。最近，我们发现了这些酶催化的两种不同方式。阐明该酶如何实现其活性构象很有趣。该酶的结构模型揭示了存在一个额外的C末端序列（ECTS）。为了解其作用，构建了一种缺失该序列的酶。然后我们比较了野生型和截短型酶的动力学参数、远紫外圆二色光谱、热稳定性、分子动力学模拟和寡聚状态。我们发现截短型酶聚集且几乎无活性，仅保留20%的总相互作用，并且与野生型酶相比，它表现出较小寡聚状态的可溶性部分。这些发现表明ECTS在维持其活性四聚体状态中起关键作用。该序列是首次在古菌丙酮酸激酶中报道，也存在于其他古菌和细菌中。丙酮酸激酶中ECTS的系统发育分析显示其分布稀疏，如果ECTS代表一个易于丢失的古老结构域，这或许可以得到解释。
