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 acyl-CoAs was investigated using both fluorimetric analysis and isothermal titration microcalorimetry, neither of which requires the physical separation of bound and free ligand for determining the dissociation constants (K(d)). The displacement of 11-(dansylamino)undecanoyl-CoA (DAUDA-CoA) from ACBP yielded binding parameters for the competing acyl-CoAs that compared favourably with those obtained using ultra-sensitive microcalorimetric titration. The K(d) values of ACBP for oleoyl-CoA and docosahexaenoyl-CoA are 0.014 and 0.016 microM, 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 microM for oleoyl-CoA and docosahexaenoyl-CoA, respectively. Given that CPT I was not only present at a much lower concentration but also has an appreciably lower affinity for acyl-CoAs than ACBP, it is proposed that CPT I is capable of interacting directly with ACBP-acyl-CoA binary complexes. This is supported by the fact that the enzyme activity correlated with the concentration of ACBP-bound acyl-CoA but not the free acyl-CoA. A transfer of acyl-CoA from ACBP-acyl-CoA binary complexes to CPT I could be a result of the enzyme inducing a conformational alteration in the ACBP leading to the release of acyl-CoA.
Abo-Hashema
K A
KA
School of Biological Sciences and Biotechnology, Division of Science and Engineering, Murdoch University, Murdoch 6150, Western Australia, Australia.
Cake
M H
MH
Lukas
M A
MA
Knudsen
J
J
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002151:Calorimetry; D002334:Carnitine O-Palmitoyltransferase / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003619:Dansyl Compounds / Q000737:chemistry / Q000378:metabolism; D026261:Diazepam Binding Inhibitor; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000214:Acyl Coenzyme A; D002352:Carrier Proteins; D003619:Dansyl Compounds; D026261:Diazepam Binding Inhibitor; D005227:Fatty Acids; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C054400:11-(dansylamino)undecanoic acid; D002334:Carnitine O-Palmitoyltransferase