Phosphopantetheine adenylyltransferase from Escherichia coli:: Investigation of the kinetic mechanism and role in regulation of coenzyme a biosynthesis
Phosphopantetheine adenylyltransferase from Escherichia coli:: Investigation of the kinetic mechanism and role in regulation of coenzyme a biosynthesis
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DOI：
10.1128/jb.00732-07
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发表时间：
2007-11-01
影响因子：
3.2
通讯作者：
Thanabal, Venkataraman
中科院分区：
文献类型：
--
作者：
Miller, J. Richard;Ohren, Jeffrey;Thanabal, Venkataraman
磷酸泛酰巯基乙胺腺苷酰转移酶（PPAT）是大肠杆菌中一种重要的六聚体酶，催化辅酶A（CoA）生物合成的倒数第二步，是抗菌药物开发的靶点。该酶利用Mg-ATP和磷酸泛酰巯基乙胺（PhP）产生脱磷酸辅酶A（dPCoA）和焦磷酸。当在E.在大肠杆菌中，PPAT与紧密结合的CoA共纯化，表明该辅因子的反馈抑制作用。使用酶偶联法的正向反应（dPCoA生成）和等温滴定量热法，我们研究了稳态动力学和配体结合性能的PPAT。所有底物和产物结合游离酶，产物抑制研究与随机双双动力学机制一致。CoA抑制PPAT并与ATP、PhP和dPCoA竞争。先前公布的结构PPAT结晶在pH 5.0显示一半的网站反应性PhP和dPCoA和ATP和CoA的全部占用。在pH 8.0的配体结合研究表明，ATP，PhP，dPCoA，和CoA占据所有六个单体的PPAT六聚体，虽然CoA表现出两种不同的结合模式。这些结果表明，在PPAT晶体结构中观察到的半位点反应性可能是pH依赖性的。鉴于以前的研究CoA生物合成的调节，PPAT动力学和配体结合数据表明，细胞内的PhP浓度调节高和低亲和力CoA结合模式之间的PPAT单体的分布。该模型与PPAT作为相对于泛酸激酶的途径通量的“备份”调节剂一致。
Phosphopantetheine adenylyltransferase (PPAT) from Escherichia coli is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and is a target for antibacterial drug discovery. The enzyme utilizes Mg-ATP and phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) and pyrophosphate. When overexpressed in E. coli, PPAT copurifies with tightly bound CoA, suggesting a feedback inhibitory role for this cofactor. Using an enzyme-coupled assay for the forward-direction reaction (dPCoA-generating) and isothermal titration calorimetry, we investigated the steady-state kinetics and ligand binding properties of PPAT. All substrates and products bind the free enzyme, and product inhibition studies are consistent with a random bi-bi kinetic mechanism. CoA inhibits PPAT and is competitive with ATP, PhP, and dPCoA. Previously published structures of PPAT crystallized at pH 5.0 show half-the-sites reactivity for PhP and dPCoA and full occupancy by ATP and CoA. Ligand-binding studies at pH 8.0 show that ATP, PhP, dPCoA, and CoA occupy all six monomers of the PPAT hexamer, although CoA exhibits two thermodynamically distinct binding modes. These results suggest that the half-the-sites reactivity observed in PPAT crystal structures may be pH dependent. In light of previous studies on the regulation of CoA biosynthesis, the PPAT kinetic and ligand binding data suggest that intracellular PhP concentrations modulate the distribution of PPAT monomers between high- and low-affinity CoA binding modes. This model is consistent with PPAT serving as a "backup" regulator of pathway flux relative to pantothenate kinase.