Steady-state kinetics of indole-3-glycerol phosphate synthase from Mycobacterium tuberculosis.
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.
Czekster
Clarissa M
CM
Centro de Pesquisas em Biologia Molecular e Funcional, Instituto de Ciência e Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, 6681/92A Ipiranga Av., 90619-900 Porto Alegre, RS, Brazil.
Neto
Brenno A D
BA
Lapis
Alexandre A M
AA
Dupont
Jairton
J
Santos
Diogenes S
DS
Basso
Luiz A
LA
D001483:Base Sequence; D055592:Biophysical Phenomena; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007209:Indole-3-Glycerol-Phosphate Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D012274:Ribulosephosphates / Q000138:chemical synthesis / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics; D014774:Virulence
D017931:DNA Primers; D004269:DNA, Bacterial; D012274:Ribulosephosphates; C022983:1-(2-carboxyphenylamino)-1-deoxyribulose 5-phosphate; D007209:Indole-3-Glycerol-Phosphate Synthase