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  • Title: Kinetics of fatty acid interactions with fatty acid binding proteins from adipocyte, heart, and intestine.
    Author: Richieri GV, Ogata RT, Kleinfeld AM.
    Journal: J Biol Chem; 1996 May 10; 271(19):11291-300. PubMed ID: 8626681.
    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.
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