CRABPs Alter all-trans-Retinoic Acid Metabolism by CYP26A1 via Protein-Protein Interactions

Cellular retinoic acid binding proteins (CRABP1 and CRABP2) bind all-trans-retinoic acid (atRA), the active metabolite of vitamin A, with high affinity. CRABP1 and CRABP2 have been shown to interact with the atRA-clearing cytochrome P450 enzymes CYP26B1 and CYP26C1 and with nuclear retinoic acid receptors (RARs). We hypothesized that CRABP1 and CRABP2 also alter atRA metabolism and clearance by CYP26A1, the third key atRA-metabolizing enzyme in the CYP26 family. Based on stopped-flow experiments, atRA bound CRABP1 and CRABP2 with K_d values of 4.7 nM and 7.6 nM, respectively. The unbound atRA K_m values for 4-OH-atRA formation by CYP26A1 were 4.7 ± 0.8 nM with atRA, 6.8 ± 1.7 nM with holo-CRABP1 and 6.1 ± 2.7 nM with holo-CRABP2 as a substrate. In comparison, the apparent k_cat value was about 30% lower (0.71 ± 0.07 min^-1 for holo-CRABP1 and 0.75 ± 0.09 min^-1 for holo-CRABP2) in the presence of CRABPs than with free atRA (1.07 ± 0.08 min^-1). In addition, increasing concentrations in apo-CRABPs decreased the 4-OH-atRA formation rates by CYP26A1. Kinetic analyses suggest that apo-CRABP1 and apo-CRABP2 inhibit CYP26A1 (K_i = 0.39 nM and 0.53 nM, respectively) and holo-CRABPs channel atRA for metabolism by CYP26A1. These data suggest that CRABPs play a critical role in modulating atRA metabolism and cellular atRA concentrations.