Influence of α-CH→NH Substitution in C₈-CoA on the Kinetics of Association and Dissociation of Ligands with Medium Chain Acyl-CoA Dehydrogenase

Abstract

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.

Acyl‐CoA dehydrogenases

“…In MCAD, Glu376 is located on loop JK, while in long chain acyl‐CoA dehydrogenase (LCAD) and isovaleryl‐CoA dehydrogenase (i3VD) a corresponding Glu is placed at position 255 on the adjacent helix G, the overall topology at the active center being conserved (see discussion in the accompanying paper [5]). A Glu376Asp mutation in MCAD shows ≈ 5% of the activity of the wild‐type (wt) form [63,64]. This difference was attributed to the nonoptimal positioning of the base in the Asp case.…”

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

“…The microcalorimetric titration data for the binding of octenoyl‐CoA to MCAD yielded the average values of n , ΔG°, ΔH°, and TΔS° to be 0.98 ± 0.06, −8.3 ± 0.2 kcal/mole, −17.2 ± 1.6 kcal/mole, and −8.9 ± 1.8 kcal/mole, respectively (Peterson et al 1998). A comparative account of these data reveals that whereas the magnitudes of n and ΔG° for the binding of 2‐azaoctanoyl‐CoA are similar to those obtained for the binding of octenoyl‐CoA, the ΔH° and TΔS° values are considerably different (Peterson et al 2000). Of the latter parameters, the ΔH° and TΔS° values for the binding of 2‐azaoctanoyl‐CoA to MCAD are 4.5 and 4.1 kcal/mole more negative, respectively, (i.e., enthalpically more favorable, but entropically less favorable) than those obtained for the binding of octenoyl‐CoA to the enzyme.…”

“…We recently undertook a comparative transient kinetic investigation for the binding of octenoyl‐CoA and 2‐azaoctanoyl‐CoA (a redox inactive ligand in which the α‐CH group is replaced by NH) via the stopped‐flow method (Peterson et al 2000). Such studies revealed that although the above substitution did not influence the overall binding affinity of the ligands to the enzyme, it influenced the microscopic steps during the overall binding process (Peterson et al 2000).…”

“…We recently undertook a comparative transient kinetic investigation for the binding of octenoyl‐CoA and 2‐azaoctanoyl‐CoA (a redox inactive ligand in which the α‐CH group is replaced by NH) via the stopped‐flow method (Peterson et al 2000). Such studies revealed that although the above substitution did not influence the overall binding affinity of the ligands to the enzyme, it influenced the microscopic steps during the overall binding process (Peterson et al 2000). The binding of these as well as other ligands to the enzyme was found to proceed via two steps (Johnson et al 1992; Kumar and Srivastava 1994,1995; Srivastava et al 1995): The first (fast) step involved the formation of the enzyme–ligand collision/Michaelis complex, followed by the isomerization of the latter complex during the second (slow) step.…”

“…The binding of these as well as other ligands to the enzyme was found to proceed via two steps (Johnson et al 1992; Kumar and Srivastava 1994,1995; Srivastava et al 1995): The first (fast) step involved the formation of the enzyme–ligand collision/Michaelis complex, followed by the isomerization of the latter complex during the second (slow) step. The latter step was found to be coupled to the protein conformational change: A comparative account of the binding studies for the above ligands led to the proposition that the electrostatic field involving the carboxyl group of Glu‐376 plays an important role in stabilizing the ground and transition state structures during the isomerization steps of the cognate enzyme–ligand complexes (Peterson et al 2000). With these features in mind, we proceeded to determine the thermodynamic consequences of α‐CH 2 → NH substitution in octanoyl‐CoA on binding of ligands to the wild‐type and E376Q mutant enzymes.…”