Phosphopantetheine Adenylyltransferase from Escherichia coli: Investigation of the Kinetic Mechanism and Role in Regulation of Coenzyme A Biosynthesis

摘要: Phosphopantetheine adenylyltransferase (PPAT) from Escherichia coli is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and a target for antibacterial drug discovery. The utilizes Mg-ATP phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) pyrophosphate. When overexpressed E. coli, PPAT copurifies with tightly bound CoA, suggesting feedback inhibitory role this cofactor. Using enzyme-coupled assay forward-direction reaction (dPCoA-generating) isothermal titration calorimetry, we investigated steady-state kinetics ligand binding properties of PPAT. All substrates products bind free enzyme, product inhibition studies are consistent random bi-bi kinetic mechanism. CoA inhibits competitive ATP, PhP, dPCoA. Previously published structures crystallized at pH 5.0 show half-the-sites reactivity PhP dPCoA full occupancy by ATP CoA. Ligand-binding 8.0 dPCoA, occupy all six monomers hexamer, although exhibits two thermodynamically distinct modes. These results suggest observed crystal may be dependent. In light previous on regulation biosynthesis, data intracellular concentrations modulate distribution between high- low-affinity This model serving as "backup" regulator pathway flux relative pantothenate kinase.