Direct detection and kinetic analysis of covalent intermediate formation in the 4-amino-4-deoxychorismate synthase catalyzed reaction.
Chorismate-utilizing enzymes catalyze diverse reactions, providing critical physiological functions unique to plants, bacteria, fungi, and some parasites. Their absence in animals makes them excellent targets for antimicrobials and herbicides. 4-Amino-4-deoxychorismate synthase (ADCS) catalyzes the first step in folate biosynthsis and shares a common core mechanism with isochorismate synthase (IS) and anthranilate synthase (AS), in which nucleophile addition at C2 initiates these reactions. Evidence was presented previously [He, Z., Stigers Lavoie, K. D., Bartlett, P. A., and Toney, M. D. (2004) J. Am. Chem. Soc. 126, 2378-2385] that K274 is the nucleophile in ADCS, implying formation of a covalent intermediate. Herein, we report the direct detection of this covalent intermediate formed in ADCS-catalyzed reactions by ESI-MS. Difference spectra show the covalent intermediate has an absorption maximum at 310 nm. This was used to study the pre-steady-state kinetics of covalent intermediate formation under various conditions. Additionally, E258 in ADCS was shown to be critical to formation of the covalent intermediate by acting as a general acid catalyst for loss of the C4 hydroxyl group. The E258A/D mutants both exhibit very low activity. Acetate is a poor chemical rescue agent for E258D but an excellent one for E258A, with a 20000-fold and 3000-fold rate increase for Gln-dependent and NH(4)(+)-dependent activities, respectively. Lastly, A213 in IS (structurally homologous to K274 in ADCS) was changed to lysine in an attempt to convert IS to an ADCS-like enzyme. HPLC studies support the formation of a covalent intermediate with this mutant.
He
Ze
Z
Department of Chemistry, Central Connecticut State University, 1615 Stanley Street, New Britain, Connecticut 06050, USA.
Toney
Michael D
MD
D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D021241:Spectrometry, Mass, Electrospray Ionization; D000637:Transaminases
D000637:Transaminases; C066599:aminodeoxychorismate synthase; D019751:Intramolecular Transferases; C058486:isochorismate synthase; D019731:Carbon-Nitrogen Ligases; D008239:Lysine