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

Abstract

We showed that the ␣-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 (␣-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 (⌬H°) and heat capacity changes (⌬C p°) for the binding of 2-azaoctanoyl-CoA (⌬H°2 98 ‫ס‬ −21.7 ± 0.8 kcal/mole, ⌬C p°‫ס‬ −0.627 ± 0.04 kcal/mole/K) to the wild-type MCAD were more negative than those obtained for the binding of octenoyl-CoA (⌬H°2 98 ‫ס‬ −17.2 ± 1.6 kcal/mole, ⌬C p°‫ס‬ −0.526 ± 0.03 kcal/mole/K). Of these, the decrease in the magnitude of ⌬C p°f or 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 ⌬H°and ⌬C p°v alues for the binding of 2-azaoctanoylCoA (⌬H°2 98 ‫ס‬ −13.3 ± 0.6 kcal/mole, ⌬C p°‫ס‬ −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 (⌬H°2 98 ‫ס‬ −13.2 ± 0.6 kcal/mole, ⌬C p°‫ס‬ −0.520 ± 0.02 kcal/mole/K). However, in neither case could the experimentally determined ⌬C p°v alues 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.

Heat Capacity Effects of Water Molecules and Ions at a Protein–DNA Interface

“…In this case structures of both wild-type and mutant enzyme are available and show no structural change other than the sequestration of water. Peterson et al 49 observe a 2 0.4 kJ mol 21 K 21 change for a conservative Gln to Glu mutation for ligand binding to acylCoA dehydrogenase, which may bind water but structural detail is unknown. The previously mentioned case of sodium binding to a cleft in thrombin, 45 which is accompanied by a large number of water molecules, gives a DC p,obs of 24.4(^0.4) kJ mol 21 K 21 .…”

β‐Oxidation of 5‐hydroxydecanoate, a Putative Blocker of Mitochondrial ATP‐Sensitive Potassium Channels

“…The X‐ray crystallographic structure of this enzyme in the absence (pdb3mdd.pdb) and presence (pdb3mde.pdb) of octenoyl‐CoA was downloaded from the Brookhaven Protein Data Bank. MCAD‐bound octenoyl‐CoA (C 8 ‐CoA) was modified to decanoyl‐CoA (C 10 ‐CoA) and energy minimisation of the enzyme‐C 10 ‐CoA complex was performed as previously described (Peterson et al 2001). A hydroxyl group was added in either the ( R )‐ or ( S )‐configuration at position C‐5 to generate a model of a MCAD‐5‐HD‐CoA complex and to predict whether the hydroxyl group could sterically hinder catalysis.…”

Thermodynamics and solvent linkage of macromolecule–ligand interactions

“…In general, he proposes “significant ΔC p effects are to be expected for any macromolecular process involving a multiplicity of cooperative weak interactions of whatever kind” [25]. Experimental examples include trapping of water due to introduction of a mutation in DNA gyrase [26], uptake of water associated with dimerization of the β-lactoglobulin dimer [27], binding of different ligands by cyclophilin [28], binding of DNA by a TATA-box binding protein [29] and others [30]. In general, experimental estimates of the effect on ΔC p per trapped water molecule range from −18 cal mol −1 K −1 to −60 ± 8 cal mol −1 K −1 [25, 31].…”