Substrate Deactivation of Phenylalanine-Sensitive 3-Deoxy-d-arabino-heptulosonate 7-Phosphate Synthase by Erythrose 4-Phosphate

• Published in: Biochemistry (Easton) 2001-12, Vol.40 (49), p.14821-14828
• Main Authors: Parker, Emily J, Bulloch, Esther M. M, Jameson, Geoffrey B, Abell, Chris
• Format: Article
• Language: English
• Subjects: 3-Deoxy-7-Phosphoheptulonate Synthase - metabolism; Binding Sites; Borohydrides - metabolism; Indicators and Reagents - metabolism; Models, Molecular; Molecular Structure; Phosphoenolpyruvate - metabolism; Protein Structure, Tertiary; Spectrometry, Mass, Electrospray Ionization; Sugar Phosphates - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010928j

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS, EC 4.1.2.15) catalyzes the condensation of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to give DAH7P via an ordered sequential mechanism. In the absence of PEP (the first substrate to bind), E4P binds covalently to the phenylalanine-sensitive DAH7PS of Escherichia coli, DAH7PS(Phe), deactivating the enzyme. Activity is restored on addition of excess PEP but not if deactivation was carried out in the presence of sodium cyanoborohydride. Electrospray mass spectrometry indicates that a single E4P is bound to the protein. These data are consistent with a slow, reversible Schiff base reaction of the aldehydic functionality of E4P with a buried lysine. Molecular modeling indicates that Lys186, a residue at the base of the substrate-binding cavity involved in hydrogen bonding with PEP, is well placed to react with E4P forming an imine linkage that is substantially protected from solvent water.