title: Shikimate-3-phosphate binds to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase.
abstract: 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the transfer of the enolpyruvyl moiety from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P). Mutagenesis and X-ray crystallography data suggest that the active site of the enzyme is in the cleft between its two globular domains; however, they have not defined which residues are responsible for substrate binding and catalysis. Here we attempt to establish the binding of the substrate S3P to the isolated N-terminal domain of EPSP synthase using a combination of NMR spectroscopy and isothermal titration calorimetry. Our experimental results indicate that there is a saturable and stable conformational change in the isolated N-terminal domain upon S3P binding and that the chemical environment of the S3P phosphorus when bound to the isolated domain is very similar to that of S3P bound to EPSP synthase. We also conclude that most of the free energy of S3P binding to EPSP synthase is contributed by the N-terminal domain.
authors: School of Molecular Biosciences and Center for NMR Spectroscopy, Washington State University, Pullman, Washington 99164-4660, USA.
authors: M E
authors: ME
authors: Stauffer
authors: J K
authors: JK
authors: Young
authors: J N
authors: JN
authors: Evans
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002151:Calorimetry; D007711:Klebsiella pneumoniae / Q000201:enzymology; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010759:Phosphorus Isotopes; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D010759:Phosphorus Isotopes; D012765:Shikimic Acid; C042786:shikimic acid-3-phosphate; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase