Energetic consequences of accommodating a bulkier ligand at the active site of medium chain acyl-CoA dehydrogenase by creating a complementary enzymesite cavity

The substitution of the C=O by the C=S group in 2-azaoctanoyl-CoA increases the volume of the ligand by 11 Angstrom (3), and the excision of a methyle ne group from Glu-376, via Glu-376 --> Asp (E376D) mutation in medium chain acyl-CoA dehydrogenase (MCAD), creates a complementary cavity of Is Angstr om (3) dimension, just opposite to the ligand's carbonyl group. We investig ated whether the newly created cavity would facilitate accommodation of the bulkier (C=O --> C=S substituted) ligand within the active site of the enz yme. To ascertain this, we determined the binding affinity and kinetics of association and dissociation of 2-azaoctanoyl-CoA and the C=O --> C=S subst ituted ligand, 2-azadithiooctanoyl-CoA, involving the wild-type and Glu-376 --> Asp mutant enzymes, The experimental data revealed that the binding of 2-azadithiooctanoyl-CoA to the wild-type enzyme was energetically unfavora ble as compared to 2-azaoctanoyl-CoA. However, such an energetic constraint was alleviated for the binding of the former ligand to the E376D mutant en zyme site. A detailed account of the free energy and enthalpic profiles for the binding of 2-azaoctanoyl-CoA and 2-azadithiooctanoyl-CoA to the wild-t ype and Glu-376 --> Asp mutant enzymes throws light on the flexibility of t he enzyme site cavity in stabilizing the ground and transition states of th e enzyme-ligand complexes.