KINETICS OF FATTY-ACID INTERACTIONS WITH FATTY-ACID-BINDING PROTEINS FROM ADIPOCYTE, HEART, AND INTESTINE

Rate constants for the interaction of fatty acids (FA) with fatty acid binding proteins (FABP) from adipocyte (A-FABP), heart (H-FABP), and intestine (I-FABP) were determined by using stopped-flow fluorometry a nd ADI-FAB, the fluorescent probe of free fatty acids (FFA), or a new FFA probe, ADIFAB2, constructed by derivatizing with acrylodan the Leu (72) --> Ala mutant of I-FABP. ADI-FAB2, because its binding affinitie s are about 10-fold greater than ADIFAB, was found to be more accurate for monitoring the kinetics of the higher affinity reactions. On- (k( on)) and off- (k(off)) rate constants were determined as a function of temperature. Our results reveal that in all cases the FA-FABP equilib rium is achieved within 2 s at 37 degrees C and within 20 s at 10 degr ees C. Off-rate constants varied by about 10-fold among the different underivatized FABPs; k(off) values were smallest for H-FABP and larges t for A-FABP, while k(on) values for these proteins generally varied b y less than 2-fold. The results show that the previously reported larg er affinities of I- and H-FABPs as compared to A-FABP are primarily a reflection of larger k(on) values for I-FABP and smaller k(off) values for H-FABP. Eyring transition state theory was used to evaluate the a ctivation thermodynamic parameters for both on- and off-reactions and the results show that in virtually all cases the rate-limiting steps a re predominately enthalpic. Activation free energies for binding to AD IFAB are generally composed of about 8 kcal/mol unfavorable enthalpy a nd about a 1 kcal/mol favorable entropic contribution. For the underiv atized FABPs the activation free energies are all about 7 +/- 0.3 kcal /mol, suggesting that the transition state for entering or leaving the binding site involves a common protein structural change. We suggest that entering or leaving the FABP binding cavity involves similar mech anisms for all 3 FABPs and may involve amino acid residues located wit hin the portal regions of these proteins.