Kinetics of fatty acid interactions with fatty acid binding proteins from adipocyte, heart, and intestine.
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.
Richieri
G V
GV
Medical Biology Institute, La Jolla, California 92037, USA.
Ogata
R T
RT
Kleinfeld
A M
AM
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
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