Abstract
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 and ADIFAB, the fluorescent probe of free fatty acids (FFA), or a new FFA probe, ADIFAB2, constructed by derivatizing with acrylodan the Leu72 --> Ala mutant of I-FABP. ADIFAB2, because its binding affinities are about 10-fold greater than ADIFAB, was found to be more accurate for monitoring the kinetics of the higher affinity reactions. On- (kappa on) and off- (kappa off) rate constants were determined as a function of temperature. Our results reveal that in all cases the FA-FABP equilibrium is achieved within 2 s at 37 degrees C and within 20 s at 10 degrees C. Off-rate constants varied by about 10-fold among the different underivatized FABPs; kappa off values were smallest for H-FABP and largest for A-FABP, while kappa on values for these proteins generally varied by less than 2-fold. The results show that the previously reported larger affinities of I- and H-FABPs as compared to A-FABP are primarily a reflection of larger kappa on values for I-FABP and smaller kappa off values for H-FABP. Eyring transition state theory was used to evaluate the activation thermodynamic parameters for both on- and off-reactions and the results show that in virtually all cases the rate-limiting steps are predominantly enthalpic. Activation free energies for binding to ADIFAB are generally composed of about 8 kcal/mol unfavorable enthalpy and about a 1 kcal/mol favorable entropic contribution. For the underivatized 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 mechanisms for all 3 FABPs and may involve amino acid residues located within the portal regions of these proteins.
Highlights
EXPERIMENTAL PROCEDURESMaterials—All measurements were done using the sodium salts of the fatty acids (FA) purchased from Nu Chek Prep, Elysian, MN, as described previously [12–14]
The results demonstrate that the kinetic basis for achieving equilibrium is different in different fatty acid binding proteins (FABP); the larger affinity of I- and H-FABPs as compared to Standard deviations of 30 and 40% were used for FABP off- and on-rate constants, respectively
How interactions with acceptor membranes might affect dissociation of unlabeled fatty acids (FA) is unclear, results comparing rate constants determined for H-FABP with varying ADIFAB and H-FABP concentrations in the present study indicate that collisions between the ADIFAB acceptor and FABPs do not affect dissociation rates
Summary
Materials—All measurements were done using the sodium salts of the FA purchased from Nu Chek Prep, Elysian, MN, as described previously [12–14]. To determine how the characteristics of the kinetic model affect the accuracy with which rate constants can be determined, time courses for transfer of FA from FABP to ADIFAB were simulated by solving Equations 1–3 with arbitrary rate constants for FABP and with the measured rate constants for ADIFAB These simulated transfers reveal, as seen for the example of oleate dissociation from I-FABP, how the concentration of the three reactants, [ADIFABb], [FFA], and [FABPb] are expected to vary with time after mixing. For larger FABP off-rate constants, the rate at which FA dissociates from FABP can exceed the rate at which FA can bind to ADIFAB, thereby uncoupling the two processes This uncoupling is most evident in the time course for [FFA] where, for large koff values, the concentration at early times increases before decaying toward equilibrium. The predominance of enthalpy applies to the equilibrium thermodynamic parameters for FA binding to FABP as determined by both van’t Hoff [14] and calorimetry measurements [27, 28], the van’t Hoff determined ⌬H are larger (more favorable) by about 3 kcal/mol
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