TY - JOUR
T1 - Direct detection and kinetic analysis of covalent intermediate formation in the 4-amino-4-deoxychorismate synthase catalyzed reaction
AU - He, Ze
AU - Toney, Michael D.
PY - 2006/4/18
Y1 - 2006/4/18
N2 - 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 NH4+-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.
AB - 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 NH4+-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.
UR - http://www.scopus.com/inward/record.url?scp=33645933904&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33645933904&partnerID=8YFLogxK
U2 - 10.1021/bi052216p
DO - 10.1021/bi052216p
M3 - Article
C2 - 16605270
AN - SCOPUS:33645933904
VL - 45
SP - 5019
EP - 5028
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 15
ER -