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°C. Circular dichroism spectra analysis revealed that loss of secondary structure of rTPI occurs around 60°C. Metal cations were not required for M. tuberculosis TPI activity. The kcat was 4.1 × 106 min-1. Importantly, the apparent Km value of M. tuberculosis rTPI for the substrate glyceraldehyde-3-phosphate is 84 μM which is sevenfold higher than the value reported for human TPI. The difference in Km 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. © 2006 Federation of European Microbiological Societies.