Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil.
PLoS One. 2013 May 6;8(5):e61918. doi: 10.1371/journal.pone.0061918. Print 2013.
Tuberculosis remains as one of the main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), shown to be essential for survival of bacilli, catalyzes the phosphoryl transfer from ATP to the carbon-3 hydroxyl group of shikimate (SKH), yielding shikimate-3-phosphate and ADP. Here we present purification to homogeneity, and oligomeric state determination of recombinant MtSK. Biochemical and biophysical data suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release. Isothermal titration calorimetry (ITC) for binding of ligands to MtSK provided thermodynamic signatures of non-covalent interactions to each process. A comparison of steady-state kinetics parameters and equilibrium dissociation constant value determined by ITC showed that ATP binding does not increase the affinity of MtSK for SKH. We suggest that MtSK would more appropriately be described as an aroL-encoded type II shikimate kinase. Our manuscript also gives thermodynamic description of SKH binding to MtSK and data for the number of protons exchanged during this bimolecular interaction. The negative value for the change in constant pressure heat capacity (ΔCp) and molecular homology model building suggest a pronounced contribution of desolvation of non-polar groups upon binary complex formation. Thermodynamic parameters were deconvoluted into hydrophobic and vibrational contributions upon MtSK:SKH binary complex formation. Data for the number of protons exchanged during this bimolecular interaction are interpreted in light of a structural model to try to propose the likely amino acid side chains that are the proton donors to bulk solvent following MtSK:SKH complex formation.
结核病仍然是全球主要的死亡原因之一，其病原体是单一的结核分枝杆菌。编码 aroK 的结核分枝杆菌莽草酸激酶（MtSK）对于细菌的存活至关重要，它催化来自 ATP 的磷酸基向莽草酸（SKH）的碳-3 位羟基的转移，生成莽草酸-3-磷酸和 ADP。在这里，我们展示了重组 MtSK 的均相纯化和寡聚状态测定。生化和生物物理数据表明，单体 MtSK 催化的化学反应遵循快速平衡随机顺序的底物结合和有序产物释放。配体与 MtSK 结合的等温滴定量热法（ITC）为每个过程提供了非共价相互作用的热力学特征。通过 ITC 确定的稳态动力学参数和平衡解离常数值的比较表明，ATP 结合不会增加 MtSK 对 SKH 的亲和力。我们认为 MtSK 更适合被描述为编码 aroL 的 II 型莽草酸激酶。我们的论文还给出了 SKH 与 MtSK 结合的热力学描述以及在这个双分子相互作用中交换质子的数量的数据。在恒压热容（ΔCp）和分子同源模型构建中，常数的负值表明在二元复合物形成时，非极性基团的去溶剂化作用有显著贡献。在 MtSK：SKH 二元复合物形成时，热力学参数被分解为疏水性和振动贡献。在考虑到结构模型的情况下，对在这个双分子相互作用中交换质子的数量进行了解释，试图提出在 MtSK：SKH 复合物形成后，可能的氨基酸侧链作为质子供体向溶剂主体提供质子。
