Kinetical Analysis of Mutant PZase Enzyme To Uncover The Pyrazinamide Resistance In Mycobacterium tuberculosis Clinical Isolates.

Mutations in the pncA gene of Mycobacterium tuberculosis, which encodes the PZase enzyme, are closely linked to pyrazinamide (PZA) resistance. Two clinical isolates, R1 and R2, which are resistant to pyrazinamide at concentrations of 100 μg/mL and 150 μg/mL, respectively, harbor multiple pncA mutations. The pncA R1 gene carries T41C, G419A, and A535G, causing Cys14Arg, Arg140His, and Ser179Gly substitutions in PZase; the pncA R2 gene has G76T, G112C, A403C, and G419A, resulting in Ala26Ser, Ala38Pro, Thr135Pro, and Arg140His changes. The relationship between the biochemical characteristics of the enzymes with pyrazinamide resistance in isolates R1 and R2 remains unclear. This paper presents the catalytic kinetics of mutant enzymes and explores their association with PZA resistance. The impact of these mutations on enzyme function was evaluated by expressing recombinant PZase in Escherichia coli, resulting in the production of proteins approximately 21 kDa in size, as confirmed by SDS-PAGE analysis. Kinetic analysis revealed reduced catalytic efficiency (kcat/KM) for the PZase-R1 and PZase-R2 variants, measured at 1.759 mM¯¹·min¯¹ and 1.500 mM¯¹·min¯¹, respectively, in comparison to the wild-type enzyme, which exhibited a value of 2.443 mM¯¹·min¯¹. These reductions correspond to decreases of 28.0% for PZase-R1 and 38.6% for PZase-R2. The observed declines in catalytic efficiency are likely to contribute to the development of resistance to pyrazinamide (PZA). Further studies, including site-directed mutagenesis and structural modelling, are needed to clarify the impact of each mutation on PZase function and drug interaction.