The interaction of acyl-CoA with acyl-CoA binding protein and carnitine palmitoyltransferase I

The affinity of recombinant rat acyl-CoA binding protein (ACBP) towards acy l-CoAs was investigated using both fluorimetric analysis and isothermal tit ration microcalorimetry, neither of which requires the physical separation of bound and free ligand for determining the dissociation constants (Kd). T he displacement of 11-(dansyl-amino)undecanoyl-CoA (DAUDA-CoA) from ACBP yi elded binding parameters for the competing acyl-CoAs that compared favourab ly with those obtained using ultra-sensitive micro calorimetric titration. The Kd values of ACBP for oleoyl-CoA and docosahexaenoyl-CoA are 0.014 and 0.016 muM, respectively. Under identical experimental conditions, carnitine palmitoyltransferase I (CPT I) of purified rat liver mitochondria has K-d values of 2.4 and 22.7 PM for oleoyl-CoA and docosahexaenoyl-CoA, respectiv ely. Given that CPT I was not only present at a much lower concentration bu t also has an appreciably lower affinity for acyl-CoAs than ACBP, it is pro posed that CPT I is capable of interacting directly with ACBP-acyl-CoA bina ry complexes. This is supported by the fact that the enzyme activity correl ated with the concentration of ACBP-bound acyl-CoA but not the free acyl-Co A. A transfer of acyl-CoA from ACBP-acyl-CoA binary complexes to CPT I coul d be a result of the enzyme inducing a conformational alteration in the ACB P leading to the release of acyl-CoA. (C) 2001 Elsevier Science Ltd. All ri ghts reserved.