Fatty acid interactions with native and mutant fatty acid binding proteins

• Published in: Molecular and cellular biochemistry Vol. 192; no. 1-2; pp. 77 - 85
• Main Authors: Richieri, G V, Ogata, R T, Kleinfeld, A M
• Format: Journal Article
• Language: English
• Published: Netherlands Springer Nature B.V 01-02-1999
• Subjects: Amino acids; Animals; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Enthalpy; Entropy; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Fatty acids; Fatty Acids - chemistry; Fatty Acids - metabolism; Fluorescent Dyes - metabolism; Kinetics; Models, Biological; Models, Molecular; Myelin P2 Protein - chemistry; Myelin P2 Protein - metabolism; Neoplasm Proteins; Nerve Tissue Proteins; Proteins; Rats; Thermodynamics; Tissue Distribution
• ISSN: 0300-8177; 1573-4919
• DOI: 10.1023/A:1006826405151

The interactions of long chain fatty acids (FA) with wild type (WT) fatty acid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constants for binding and dissociation. These measurements have been done using the fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of the free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that forWT proteins from adipocyte, heart, intestine, and liver, Kd values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Binding affinities for heart and liver are generally greater than those for adipocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37 degrees C is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FFA, suggests a buffering action of FABPs that helps to maintain the intracellular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature dependence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of the intestinal FABP. These measurements showed that binding affinities did not report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an alanine substitution for arginine 106 yields a 30 fold increase in binding affinity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg106, is more than compensated for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.