Kinetics of Fatty Acid Interactions with Fatty Acid Binding Proteins from Adipocyte, Heart, and Intestine

Gary V. Richieri, Ronald T. Ogata, Alan M. Kleinfeld
1996 Journal of Biological Chemistry  
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 ADI-FAB, the fluorescent probe of free fatty acids (FFA), or a new FFA probe, ADIFAB2, constructed by derivatizing with acrylodan the Leu 72 3 Ala mutant of I-FABP. ADI-FAB2, because its binding affinities are about 10-fold greater than ADIFAB, was found to be more accurate for
more » ... ng 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 equilibrium is achieved within 2 s at 37°C and within 20 s at 10°C. Off-rate constants varied by about 10-fold among the different underivatized FABPs; k off values were smallest for H-FABP and largest for A-FABP, while k 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 k on values for I-FABP and smaller k 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 vir- tually all cases the rate-limiting steps are predominately 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.
doi:10.1074/jbc.271.19.11291 pmid:8626681 fatcat:4qgngzlnx5gezabaplsyfrcdom