RESUMEN

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.

Asunto(s)

Indol-3-Glicerolfosfato Sintasa/metabolismo , Mycobacterium tuberculosis/enzimología , Secuencia de Bases , Fenómenos Biofísicos , Clonación Molecular , Cartilla de ADN/genética , ADN Bacteriano/genética , Genes Bacterianos , Concentración de Iones de Hidrógeno , Indol-3-Glicerolfosfato Sintasa/genética , Indol-3-Glicerolfosfato Sintasa/aislamiento & purificación , Cinética , Espectroscopía de Resonancia Magnética , Modelos Químicos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Ribulosafosfatos/síntesis química , Ribulosafosfatos/metabolismo , Espectrometría de Fluorescencia , Espectrometría de Masa por Ionización de Electrospray , Termodinámica , Virulencia