FK506-BINDING PROTEIN MUTATIONAL ANALYSIS - DEFINING THE ACTIVE-SITE RESIDUE CONTRIBUTIONS TO CATALYSIS AND THE STABILITY OF LIGAND COMPLEXES

The 12 kDa FK506-binding protein FKBP12 is a cis-trans peptidyl-prolyl isomerase that binds the macrolides FK506 and rapamycin, We have exam ined the role of the binding pocket residues of FKBP12 in protein-liga nd interactions by making conservative substitutions of 12 of these re sidues by site-directed mutagenesis. For each mutant FKBP12, we measur ed the affinity for FK506 and rapamycin and the catalytic efficiency i n the cis-trans peptidyl-prolyl isomerase reaction, The mutation of Tr p59 or Phe99 generates an FKBP12 with a significantly lower affinity f or FK506 than wild-type protein, Tyr26 and Tyr82 mutants are enzymatic ally active, demonstrating that hydrogen bonding by these residues is not required for catalysis of the cis-trans peptidyl-prolyl isomerase reaction, although these mutations alter the substrate specificity of the enzyme, We conclude that hydrophobic interactions in the active si te dominate in the stabilization of FKBP12 binding to macrolide ligand s and to the twisted-amide peptidyl-prolyl substrate intermediate.