title: Kinetics of fatty acid interactions with fatty acid binding proteins from adipocyte, heart, and intestine.
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.
authors.affiliation: Medical Biology Institute, La Jolla, California 92037, USA.
authors.forename: G V
authors.initials: GV
authors.lastname: Richieri
authors.forename: R T
authors.initials: RT
authors.lastname: Ogata
authors.forename: A M
authors.initials: AM
authors.lastname: Kleinfeld
mesh_terms: D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D005230:Fatty Acids, Nonesterified / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine; D008433:Mathematics; D051379:Mice; D008962:Models, Theoretical; D018996:Myelin P2 Protein / Q000737:chemistry / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D025521:Tumor Suppressor Proteins
chemical_list: D002352:Carrier Proteins; C107608:FABP7 protein, human; C082667:Fabp5 protein, mouse; C086936:Fabp7 protein, mouse; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D005230:Fatty Acids, Nonesterified; D005456:Fluorescent Dyes; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009829:Oleic Acids; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; C102579:acrylodated intestinal fatty acid-binding protein 2; D019301:Oleic Acid; D007930:Leucine