AbstractThe cellular milieu is a complex and crowded aqueous solution. Macromolecular crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects, if any, of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (K _m , k _cat , and k _cat /K _m ) of Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase (InhA). Negligible effects on InhA activity were observed for ficoll 70, ficoll 400 and dextran 70. A complex effect was observed for PEG 6000. Glucose and sucrose showed, respectively, no effect on InhA activity and decreased k _cat /K _m for NADH and k _cat for 2-trans-dodecenoyl-CoA. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution. The effects of the crowding agents on the energy (E _a and E _η ), enthalpy (∆H ^# ), entropy (∆S ^# ), and Gibbs free energy (∆G ^# ) of activation were determined. The ∆G ^# values for all crowding agents were similar to buffer, suggesting that excluded volume effects did not facilitate stable activated ES ^# complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that “soft” interactions play a role in crowding effects. The results on InhA do not unequivocally meet the criteria for crowding effect due to exclude volume only.