A conserved lysine residue (Lys69) in Mycobacterium tuberculosis shikimate dehydrogenase (MtbSD) plays a catalytic role but not in substrate binding. The study used site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements, and molecular modeling to investigate the role of Lys69 in MtbSD. The wild-type MtbSD had a catalytic constant (k_cat) of 50 s⁻¹, while the K69A mutant had a much lower value of 0.73 s⁻¹, indicating that Lys69 is essential for catalysis. The K_m values for DHS and NADPH increased in the mutant, but the mutant still bound these substrates with only slightly higher K_m values than the wild-type enzyme, suggesting proper folding. Equilibrium dissociation constants for wild-type and K69A mutant enzymes were 32 (±4) μM and 134 (±21) μM, respectively. Structural analysis showed that the K69A model had a low RMSD deviation from the template, indicating that the mutant protein is properly folded. The Lys69 residue is likely involved in stabilizing the developing negative charge at the hydride-accepting C-3 carbonyl oxygen of DHS during the forward reaction, acting as an acid-base catalytic group. The results suggest that Lys69 is critical for catalysis but not for substrate binding. These findings may help in the rational design of specific inhibitors targeting MtbSD, which could lead to the development of new antitubercular drugs.A conserved lysine residue (Lys69) in Mycobacterium tuberculosis shikimate dehydrogenase (MtbSD) plays a catalytic role but not in substrate binding. The study used site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements, and molecular modeling to investigate the role of Lys69 in MtbSD. The wild-type MtbSD had a catalytic constant (k_cat) of 50 s⁻¹, while the K69A mutant had a much lower value of 0.73 s⁻¹, indicating that Lys69 is essential for catalysis. The K_m values for DHS and NADPH increased in the mutant, but the mutant still bound these substrates with only slightly higher K_m values than the wild-type enzyme, suggesting proper folding. Equilibrium dissociation constants for wild-type and K69A mutant enzymes were 32 (±4) μM and 134 (±21) μM, respectively. Structural analysis showed that the K69A model had a low RMSD deviation from the template, indicating that the mutant protein is properly folded. The Lys69 residue is likely involved in stabilizing the developing negative charge at the hydride-accepting C-3 carbonyl oxygen of DHS during the forward reaction, acting as an acid-base catalytic group. The results suggest that Lys69 is critical for catalysis but not for substrate binding. These findings may help in the rational design of specific inhibitors targeting MtbSD, which could lead to the development of new antitubercular drugs.