Binding kinetics of engineered mutants provide insight about the pathway for entering and exiting the intestinal fatty acid binding protein

To better understand the mechanism by which fatty acids bind to and dissoci ate from the binding cavities of fatty acid binding proteins (FABPs), we co nstructed 31 single amino acid mutants of the intestinal FABP (I-FABP) and determined the rate constants for binding and dissociation, primarily for l ong-chain fatty acids (FA). FA dissociation from these proteins was measure d both by the ADIFAB method and by the change in tryptophan fluorescence of the FABPs. Rate constants for binding (k(on)) were calculated from the rat e constants for dissociation (k(off)) and the equilibrium binding affinitie s. Amino acid substitutions were made at locations within the binding cavit y, in the region of the gap between the beta D- and beta E-strands, and wit hin the "portal" region of the protein. The k(off) values for the mutant pr oteins ranged from about 20-fold slower to 4-fold faster than the wild-type (WT) protein. Values for k(on) were as much as 20-fold slower than the WT protein, but in no case was k(on) significantly faster than the WT. Mutants with slower and faster k(off) values were generally those involving sites within the binding cavity and, relative to the WT protein, revealed higher and lower affinities, respectively. Reduced rates of binding were generally , but, not exclusively, associated with sites within the portal region. For example, for F68A which is located closer to the opposite end of the prote in from the portal region, the k(on) is more than 10-fold slower than WT. E ven for these distal sites, however, the evidence is consistent with reduct ions in k(on) being due to alterations of the portal region. Binding affini ties and rate constants measured as a function of ionic strength also sugge st that the FA initially binds, through an electrostatic interaction, to Ar g-56 on the surface of the protein, before inserting into the binding cavit y. Thus, the results of this study are consistent with FA binding to I-FABP involving an initial interaction with Arg-56 followed by insertion of the FA, through the portal region, into the binding cavity and with a reversal of these steps for the dissociation reaction.