Liver fatty acid binding protein FABP1 transfers substrates to cytochrome P450 4A11 for catalysis.

Cytochrome P450 (P450) 4A11 is a human P450 family 4 ω-oxidase that selectively catalyzes the hydroxylation of the terminal methyl group of fatty acids. Cytosolic lipids are the substrates for the enzyme but are considered to be primarily bound in cells by liver fatty acid binding protein (FABP1). Lipid binding to recombinant FABP1 with a fluorophore displacement assay showed substantial preference of FABP1 for ≥16-carbon fatty acids (K < 70 nM). Comparison of palmitate-binding studies revealed that FABP1 bound the lipid >100-fold more tightly than P450 4A11. Tight binding of P450 4A11 to Alexa-488 dye-labeled FABP1 was observed in fluorescence assays, and the interaction was dependent on ionic strength (K = 3-124 nM). Kinetic studies with Alexa-FABP1 indicated that the rate of protein-protein association is fast (∼2 s), and a palmitate delivery experiment suggested that substrate transfer (from FABP1 to P450) is not rate limiting. From these results, we constructed a kinetic model of the FABP1-P450 interaction and applied it to a catalytic study of FABP1 on P450 4A11 palmitate ω-hydroxylation, the results of which conclusively rejected the free ligand hypothesis. Our results are explained by a direct transfer model in which lipid-bound FABP1 interacts with P450 4A11, transfers the substrate, and a slower P450 conformational change follows to position the molecule in a mode for oxidation. Given the limited free lipid pool in vivo, interaction with FABP1 may be a dominant mechanism by which P450 4A11 accesses its substrates and may offer a novel means to target P450 4A11 activity.