title: Biochemical and functional characterization of triosephosphate isomerase from Mycobacterium tuberculosis H37Rv.
abstract: Triosephosphate isomerase (TPI), one of the key enzymes of the glycolytic pathway, is an attractive drug target against Mycobacterium tuberculosis as glycolysis provides the majority of the organism's energy requirements inside macrophages. To carry out biochemical and biophysical characterization, purified recombinant M. tuberculosis TPI produced in Escherichia coli was used. Mass spectrum analysis showed M. tuberculosis rTPI to be of 28 213 Da. The biologically active enzyme is a homodimer as determined by gel filtration chromatography. The M. tuberculosis TPI had a pH optimum in the range of 6-8 and a temperature optimum around 37 degrees C. Circular dichroism spectra analysis revealed that loss of secondary structure of rTPI occurs around 60 degrees C. Metal cations were not required for M. tuberculosis TPI activity. The k(cat) was 4.1 x 10(6) min(-1). Importantly, the apparent K(m) value of M. tuberculosis rTPI for the substrate glyceraldehyde-3-phosphate is 84 microM which is sevenfold higher than the value reported for human TPI. The difference in K(m) is indicative of the difference in the active site of the human and M. tuberculosis TPI, which can be exploited for drug designing specifically targeting M. tuberculosis TPI.
authors.affiliation: Gene Regulation Laboratory, National Institute of Immunology, New Delhi, India.
authors.forename: Divya
authors.initials: D
authors.lastname: Mathur
authors.forename: Gunjan
authors.initials: G
authors.lastname: Malik
authors.forename: Lalit C
authors.initials: LC
authors.lastname: Garg
mesh_terms: D004269:DNA, Bacterial / Q000032:analysis; D005798:Genes, Bacterial; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014305:Triose-Phosphate Isomerase / Q000032:analysis* / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D014305:Triose-Phosphate Isomerase