TY - JOUR
T1 - Steady-state kinetics of indole-3-glycerol phosphate synthase from Mycobacterium tuberculosis
AU - Czekster, Clarissa M.
AU - Neto, Brenno A.D.
AU - Lapis, Alexandre A.M.
AU - Dupont, Jairton
AU - Santos, Diogenes S.
AU - Basso, Luiz A.
PY - 2009/6/1
Y1 - 2009/6/1
N2 - Indole-3-glycerol phosphate synthase (IGPS) catalyzes the irreversible ring closure of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), through decarboxylation and dehydration steps, releasing indole-3-glycerol phosphate (IGP), the fourth step in the biosynthesis of tryptophan. This pathway is essential for Mycobacterium tuberculosis virulence. Here we describe the cloning, expression, purification, and kinetic characterization of IGPS from M. tuberculosis. To perform kinetic studies, CdRP was chemically synthesized, purified, and spectroscopically and spectrometrically characterized. CdRP fluorescence was pH-dependent, probably owing to excited-state intramolecular proton transfer. The activation energy was calculated, and solvent isotope effects and proton inventory studies were performed. pH-rate profiles were carried out to probe for acid/base catalysis, showing that a deprotonated residue is necessary for CdRP binding and conversion to IGP. A model to describe a steady-state kinetic sequence for MtIGPS-catalized chemical reaction is proposed.
AB - Indole-3-glycerol phosphate synthase (IGPS) catalyzes the irreversible ring closure of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), through decarboxylation and dehydration steps, releasing indole-3-glycerol phosphate (IGP), the fourth step in the biosynthesis of tryptophan. This pathway is essential for Mycobacterium tuberculosis virulence. Here we describe the cloning, expression, purification, and kinetic characterization of IGPS from M. tuberculosis. To perform kinetic studies, CdRP was chemically synthesized, purified, and spectroscopically and spectrometrically characterized. CdRP fluorescence was pH-dependent, probably owing to excited-state intramolecular proton transfer. The activation energy was calculated, and solvent isotope effects and proton inventory studies were performed. pH-rate profiles were carried out to probe for acid/base catalysis, showing that a deprotonated residue is necessary for CdRP binding and conversion to IGP. A model to describe a steady-state kinetic sequence for MtIGPS-catalized chemical reaction is proposed.
KW - 1-(o-Carboxyphenylamino)-1-deoxyribulose 5-phosphate
KW - Drug target
KW - IGPS
KW - Indole-3-glycerol phosphate synthase
KW - Steady-state kinetics
KW - Tuberculosis
UR - https://www.scopus.com/pages/publications/67349108325
U2 - 10.1016/j.abb.2009.04.001
DO - 10.1016/j.abb.2009.04.001
M3 - Article
C2 - 19364491
AN - SCOPUS:67349108325
SN - 0003-9861
VL - 486
SP - 19
EP - 26
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -