Influence of alpha-CH -> NH substitution in C-8-CoA on the kinetics of association and dissociation of ligands with medium chain acyl-CoA dehydrogenase

We previously reported that the kinetic profiles for the association and di ssociation of functionally diverse C-8-CoA-ligands, viz., octanoyl-CoA (sub strate), octenoyl-CoA (product), and octynoyl-CoA (inactivator) with medium chain acyl-CoA dehydrogenase (MCAD), were essentially identical, suggestin g that the protein conformational changes played an essential role during l igand 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 rapi d-scanning and single wavelength stopped-flow data for the binding of 2-aza octanoyl-CoA to MCAD revealed that the overall interaction proceeds via two steps. The first (fast) step involves the formation of an enzyme-ligand co llision complex (with a dissociation constant of K,), followed by a slow is omerization 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 s tep is coupled to the changes in the protein conformation. Although the ove rall binding affinity (Kd) Of the enzyme-2-azaoctanoyl-CoA complex is simil ar to that of the enzyme-octenoyl-CoA complex, their microscopic equilibria within the collision and isomerized complexes show an opposite relationshi p. These results coupled with the isothermal titration microcalorimetric st udies lead to the suggestion that the electrostatic interaction within the enzyme site phase modulates the microscopic steps, as well as their corresp onding ground and transition states, during the course of the enzyme-ligand interaction.