Abstract
The glpX gene encodes for the Class II fructose-1,6-bisphosphatase enzyme in Mycobacterium tuberculosis (Mt), an essential enzyme for pathogenesis. We have performed site directed mutagenesis to introduce two mutations at residue Thr84, T84A and T84S, to explore the binding affinity of the substrate and the catalytic mechanism. The T84A mutant fully abolishes enzyme activity while retaining substrate binding affinity. In contrast, the T84S mutant retains some activity having a 10 times reduction in Vmax and exhibited similar sensitivity to lithium when compared to the wildtype. Homology modeling using the Escherichia coli enzyme structure suggests that the replacement of the critical nucleophile OH- in the Thr84 residue of the wildtype of MtFBPase by Ser84 results in subtle alterations of the position and orientation that reduce the catalytic efficiency. This mutant could be used to trap reaction intermediates, through crystallographic methods, facilitating the design of potent inhibitors via structure-based drug design.