The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase (MtbSD). The study investigates the kinetic properties of wild-type and K69A mutant enzymes to understand the role of Lysine69. Site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements, and molecular modeling were performed. The results show that the catalytic constant for the wild-type enzyme is 68-fold higher than that for the mutant, indicating that Lysine69 is crucial for catalysis but not for substrate binding. The equilibrium dissociation constants for the wild-type and mutant enzymes are 32 μM and 134 μM, respectively. Structural analysis using comparative homology modeling suggests that Lysine69 is likely involved in stabilizing the developing negative charge at the hydride-accepting C-3 carbonyl oxygen of DHS during the reduction reaction. These findings provide insights into the molecular basis of DHS/SHK recognition and catalysis, which could aid in the design of specific inhibitors for the development of antitubercular drugs.The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase (MtbSD). The study investigates the kinetic properties of wild-type and K69A mutant enzymes to understand the role of Lysine69. Site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements, and molecular modeling were performed. The results show that the catalytic constant for the wild-type enzyme is 68-fold higher than that for the mutant, indicating that Lysine69 is crucial for catalysis but not for substrate binding. The equilibrium dissociation constants for the wild-type and mutant enzymes are 32 μM and 134 μM, respectively. Structural analysis using comparative homology modeling suggests that Lysine69 is likely involved in stabilizing the developing negative charge at the hydride-accepting C-3 carbonyl oxygen of DHS during the reduction reaction. These findings provide insights into the molecular basis of DHS/SHK recognition and catalysis, which could aid in the design of specific inhibitors for the development of antitubercular drugs.