Energetic consequences of accommodating a bulkier ligand at the active site of medium chain acyl-CoA dehydrogenase by creating a complementary enzyme site cavity |
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Authors: | Peterson K M Srivastava D K |
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Affiliation: | Department of Biochemistry and Molecular Biology, North Dakota State University, Fargo, North Dakota 58105, USA. |
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Abstract: | The substitution of the C=O by the C=S group in 2-azaoctanoyl-CoA increases the volume of the ligand by 11 A(3), and the excision of a methylene group from Glu-376, via Glu-376 --> Asp (E376D) mutation in medium chain acyl-CoA dehydrogenase (MCAD), creates a complementary cavity of 18 A(3) dimension, just opposite to the ligand's carbonyl group. We investigated whether the newly created cavity would facilitate accommodation of the bulkier (C=O --> C=S substituted) ligand within the active site of the enzyme. To ascertain this, we determined the binding affinity and kinetics of association and dissociation of 2-azaoctanoyl-CoA and the C=O --> C=S substituted 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 unfavorable as compared to 2-azaoctanoyl-CoA. However, such an energetic constraint was alleviated for the binding of the former ligand to the E376D mutant enzyme 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-type and Glu-376 --> Asp mutant enzymes throws light on the flexibility of the enzyme site cavity in stabilizing the ground and transition states of the enzyme-ligand complexes. |
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