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Influence of Glu‐376 → Gln mutation on enthalpy and heat capacity changes for the binding of slightly altered ligands to medium chain acyl‐CoA dehydrogenase
Author(s) -
Peterson Karen M.,
Gopalan K.V.,
Nandy Andreas,
Srivastava D.K.
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.51401
Subject(s) - chemistry , ligand (biochemistry) , enthalpy , mole , enzyme , stereochemistry , dehydrogenase , allosteric regulation , biochemistry , thermodynamics , receptor , physics
We showed that the α‐CH 2 → NH substitution in octanoyl‐CoA alters the ground and transition state energies for the binding of the CoA ligands to medium‐chain acyl‐CoA dehydrogenase (MCAD), and such an effect is caused by a small electrostatic difference between the ligands. To ascertain the extent that the electrostatic contribution of the ligand structure and/or the enzyme site environment modulates the thermodynamics of the enzyme–ligand interaction, we undertook comparative microcalorimetric studies for the binding of 2‐azaoctanoyl‐CoA (α‐CH 2 → NH substituted octanoyl‐CoA) and octenoyl‐CoA to the wild‐type and Glu‐376 → Gln mutant enzymes. The experimental data revealed that both enthalpy (ΔH°) and heat capacity changes (ΔC p °) for the binding of 2‐azaoctanoyl‐CoA (ΔH° 298 = −21.7 ± 0.8 kcal/mole, ΔC p ° = −0.627 ± 0.04 kcal/mole/K) to the wild‐type MCAD were more negative than those obtained for the binding of octenoyl‐CoA (ΔH° 298 = −17.2 ± 1.6 kcal/mole, ΔC p ° = −0.526 ± 0.03 kcal/mole/K). Of these, the decrease in the magnitude of ΔC p ° for the binding of 2‐azaoctanoyl‐CoA (vis‐à‐vis octenoyl‐CoA) to the enzyme was unexpected, because the former ligand could be envisaged to be more polar than the latter. To our further surprise, the ligand‐dependent discrimination in the above parameters was completely abolished on Glu‐376 → Gln mutation of the enzyme. Both ΔH° and ΔC p ° values for the binding of 2‐azaoctanoyl‐CoA (ΔH° 298 = −13.3 ± 0.6 kcal/mole, ΔC p ° = −0.511 ± 0.03 kcal/mole/K) to the E376Q mutant enzyme were found to be correspondingly identical to those obtained for the binding of octenoyl‐CoA (ΔH° 298 = −13.2 ± 0.6 kcal/mole, ΔC p ° = −0.520 ± 0.02 kcal/mole/K). However, in neither case could the experimentally determined ΔC p ° values be predicted on the basis of the changes in the water accessible surface areas of the enzyme and ligand species. Arguments are presented that the origin of the above thermodynamic differences lies in solvent reorganization and water‐mediated electrostatic interaction between ligands and enzyme site groups, and such interactions are intrinsic to the molecular basis of the enzyme–ligand complementarity.