Influence of Glu-376 -> Gln mutation on enthalpy and heat capacity changesfor the binding of slightly altered ligands to medium chain acyl-CoA dehydrogenase

We showed that the alpha -CH2 --> NH substitution in octanoyl-CoA alters th e ground and transition state energies for the binding of the CoA ligands t o medium-chain acyl-CoA dehydrogenase (MCAD), and such an effect is caused by a small electrostatic difference between the ligands. To ascertain the e xtent that the electrostatic contribution of the ligand structure and/or th e enzyme site environment modulates the thermodynamics of the enzyme-ligand interaction, we undertook comparative microcalorimetric studies for the bi nding of 2-azaoctanoyl-CoA (alpha -CH2 --> NH substituted octanoyl-CoA) and octenoyl-CoA to the wild-type and Glu-376 --> Gln mutant enzymes. The expe rimental data revealed that both enthalpy (DeltaH degrees) and heat capacit y changes (DeltaC(p)degrees) for the binding of 2-azaoctanoyl-CoA (DeltaH d egrees (298) = -21.7 +/- 0.8 kcal/mole, DeltaC(p)degrees -0.627 +/- 0.04 kc al/mole/K) to the wild-type MCAD were more negative than those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -17.2 +/- 1.6 kcal/mol e, DeltaC(p)degrees = 0.526 +/- 0.03 kcal/mole/K). Of these, the decrease i n the magnitude of DeltaC(p)degrees for the binding of 2-azaoctanoyl-CoA (v is-a-vis octenoyl-CoA) to the enzyme was unexpected, because the former lig and could be envisaged to be more polar than the latter. To our further sur prise, the ligand-dependent discrimination in the above parameters was comp letely abolished on Glu-376 --> Gln mutation of the enzyme. Both DeltaH deg rees and DeltaC(p)degrees values for the binding of 2-azaoctanoyl-CoA (Delt aH degrees (298) = -13.3 +/- 0.6 kcal/mole, DeltaC(p)degrees = -0.511 +/- 0 .03 kcal/mole/K) to the E376Q mutant enzyme were found to be correspondingl y identical to those obtained for the binding of octenoyl-CoA (DeltaH degre es (298) = -13.2 +/- 0.6 kcal/mole, DeltaC(p)degrees = 0.520 +/- 0.02 kcal/ mole/K). However, in neither case could the experi mentally determined Delt aC(p)degrees values be predicted on the basis of the changes in the water a ccessible surface areas of the enzyme and ligand species. Arguments are pre sented that the origin of the above thermodynamic differences lies in solve nt reorganization and water-mediated electrostatic interaction between liga nds and enzyme site groups, and such interactions are intrinsic to the mole cular basis of the enzyme-ligand complementarity.