Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
PLoS One. 2011 Jan 11;6(1):e15228. doi: 10.1371/journal.pone.0015228.
Dephosphocoenzyme A kinase performs the transfer of the γ-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5-10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding. We show that the mycobacterial enzyme requires the Mg(2+) for its catalytic activity. The binding energetics of the interactions of the mutant enzymes with the substrates were characterized in terms of their enthalpic and entropic contributions by ITC, providing a complete picture of the effects of the mutations on activity. The properties of mutants defective in substrate recognition were consistent with the ordered sequential mechanism of substrate addition for CoaE.
脱磷酸辅酶 A 激酶将 ATP 的 γ-磷酸基团转移到脱磷酸辅酶 A 上，催化辅酶 A 生物合成的最后一步。该酶属于 P 环含 NTP 水解酶超家族，所有成员都具有由三个结构域组成的拓扑结构，包括结合受体底物的辅酶 A 结构域、核苷酸结合结构域和盖结构域。人类和分枝杆菌对应物之间在酶组织和调节方面的差异表明，结核 CoaE 是一个高可信度的药物靶点（HAMAP 数据库）。不幸的是，该酶的三维晶体结构，无论是单独的还是与任何其底物/调节剂形成复合物的结构，都没有被解析，这使得反应机制和参与底物结合、稳定和催化的主要参与者都未知。基于同源建模和序列分析，我们选择了酶的三个功能结构域中的残基，使用定点突变来评估它们对配体结合和催化的贡献。系统地突变 P 环和核苷酸结合位点的残基，鉴定了 ATP 结合中的 Lys14 和 Arg140 以及磷酸转移反应中磷酸化中间产物的稳定。突变 Asp32 和 Arg140 显示出低于野生型 5-10%的催化效率，表明这些残基在催化中起着关键作用。非保守取代 Leu114 残基，确定该亮氨酸是参与引导底物 DCoA 结合的疏水性裂缝中的关键残基。我们表明，分枝杆菌酶需要 Mg(2+) 才能发挥其催化活性。通过 ITC 从焓和熵贡献方面表征了突变酶与底物相互作用的结合能，为突变对活性的影响提供了完整的描述。在底物识别方面有缺陷的突变体的性质与 CoaE 的顺序顺序底物添加机制一致。
