Thermodynamic and kinetic properties of fatty acid interactions with rat liver fatty acid-binding protein

Fatty acid-binding protein from rat liver (L-FABP) binds 2 fatty acids (FA) per protein, in contrast to FABPs from adipocyte, heart, and intestine, for which binding and structural studies are consistent with a single FA binding site, To understand better the unique characteristics of L-FABP, we have carried out equilibrium binding and kinetic measurements of long- chain FA us ing the fluorescent probes of free fatty acids (FFA), ADIFAB and ADIFAB2, to monitor the concentration of FFA in the reaction of FA with L-FABP, We found that the dissociation constants (K-d) ranged from about 1 nM to 4 mu M, being largest for myristate at 45 degrees C and smallest for oleate at 10 degrees C, and that 2 FA were bound per L-FABP for all temperatures and Fk The binding measurements also revealed that at temperatures below 37 degrees C, affinities for the two binding sites differ by between 5- and 20-fold but as the temperature was increased, the affinities converge toward equal values, Off-rate constants (K-off were similar for all FA and for temperatures between 10 and 45 degrees C, ranged from about 0.1 s(-1) to 50 s(-1). Moreover, for all FA, k(off) values for dissociation from both the high and low affinity sites were similar, indicating that binding affinity differences at the lower temperatures reflect lower on-rates for binding to the ion: affinity site, The temperature at which the affinities of the two sites become equivalent depends upon the FA; higher temperatures (45-50 degrees C) are required for the unsaturated FA and myristate than for the longer chain saturated FA (<37 degrees C. This transition from different to equivalent affinity binding sites at specific tempera tures reflects a nonlinear van't Hoff behavior of the high affinity site, which in turn is a reflection of large heat capacity changes (between -0.6 and -1.2 kcal K-1 mol(-1)) that accompany FA binding to the high affinity site, These heat capacity changes, which are unique to L-FABP, do not appear to be correlated with a significant conformational change upon ligand binding, The differences between long chain saturated and unsaturated FA suggest that the conformation of FA bound to L-FABP may differ with both FA type and temperature, and that, in comparison to other FABPs, L-FABP may have distinctly different effects on saturated and unsaturated FA metabolism.