5‐Hydroxydecanoate (5‐HD) inhibits ischaemic and pharmacological preconditioning of the heart. Since 5‐HD is thought to inhibit specifically the putative mitochondrial ATP‐sensitive K+ (KATP) channel, this channel has been inferred to be a mediator of preconditioning. However, it has recently been shown that 5‐HD is a substrate for acyl‐CoA synthetase, the mitochondrial enzyme which ‘activates’ fatty acids. Here, we tested whether activated 5‐HD, 5‐hydroxydecanoyl‐CoA (5‐HD‐CoA), is a substrate for medium‐chain acyl‐CoA dehydrogenase (MCAD), the committed step of the mitochondrial β‐oxidation pathway. Using a molecular model, we predicted that the hydroxyl group on the acyl tail of 5‐HD‐CoA would not sterically hinder the active site of MCAD. Indeed, we found that 5‐HD‐CoA was a substrate for purified human liver MCAD with a Km of 12.8 ± 0.6 μm and a kcat of 14.1 s−1. For comparison, with decanoyl‐CoA (Km∼3 μm) as substrate, kcat was 6.4 s−1. 5‐HD‐CoA was also a substrate for purified pig kidney MCAD. We next tested whether the reaction product, 5‐hydroxydecenoyl‐CoA (5‐HD‐enoyl‐CoA), was a substrate for enoyl‐CoA hydratase, the second enzyme of the β‐oxidation pathway. Similar to decenoyl‐CoA, purified 5‐HD‐enoyl‐CoA was also a substrate for the hydratase reaction. In conclusion, we have shown that 5‐HD is metabolised at least as far as the third enzyme of the β‐oxidation pathway. Our results open the possibility that β‐oxidation of 5‐HD or metabolic intermediates of 5‐HD may be responsible for the inhibitory effects of 5‐HD on preconditioning of the heart.

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We previously reported that the kinetic profiles for the association and dissociation of functionally diverse C(8)-CoA-ligands, viz., octanoyl-CoA (substrate), octenoyl-CoA (product), and octynoyl-CoA (inactivator) with medium chain acyl-CoA dehydrogenase (MCAD), were essentially identical, suggesting that the protein conformational changes played an essential role during ligand binding and/or catalysis [Peterson, K. L., Sergienko, E. E., Wu, Y., Kumar, N. R., Strauss, A. W., Oleson, A. E., Muhonen, W. W., Shabb, J. B., and Srivastava, D. K. (1995) Biochemisry 34, 14942-14953]. To ascertain the structural basis of the above similarity, we investigated the kinetics of association and dissociation of alpha-CH-->NH-substituted C(8)-CoA, namely, 2-azaoctanoyl-CoA, with the recombinant form of human liver MCAD. The rapid-scanning and single wavelength stopped-flow data for the binding of 2-azaoctanoyl-CoA to MCAD revealed that the overall interaction proceeds via two steps. The first (fast) step involves the formation of an enzyme-ligand collision complex (with a dissociation constant of K(c)), followed by a slow isomerization step (with forward and reverse rate constants of k(f) and k(r), respectively) with concomitant changes in the electronic structure of the enzyme-bound FAD. Since the latter step involves a concurrent change in the enzyme's tryptophan fluorescence, it is suggested that the isomerization step is coupled to the changes in the protein conformation. Although the overall binding affinity (K(d)) of the enzyme-2-azaoctanoyl-CoA complex is similar to that of the enzyme-octenoyl-CoA complex, their microscopic equilibria within the collision and isomerized complexes show an opposite relationship. These results coupled with the isothermal titration microcalorimetric studies lead to the suggestion that the electrostatic interaction within the enzyme site phase modulates the microscopic steps, as well as their corresponding ground and transition states, during the course of the enzyme-ligand interaction.

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.

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