Energetic consequences of accommodating a bulkier ligand at the active site of medium chain acyl-CoA dehydrogenase by creating a complementary enzyme site cavity.
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.
Peterson
K M
KM
Department of Biochemistry and Molecular Biology, North Dakota State University, Fargo, North Dakota 58105, USA.
Srivastava
D K
DK
D000214:Acyl Coenzyme A / Q000737:chemistry; D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013552:Swine; D013696:Temperature; D013816:Thermodynamics; D017186:Titrimetry
D000214:Acyl Coenzyme A; D008024:Ligands; C034681:octanoyl-coenzyme A; D001224:Aspartic Acid; D018698:Glutamic Acid; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase