CATALYTIC AND LIGAND-BINDING PROPERTIES OF THE FK506 BINDING-PROTEIN FKBP12 - EFFECTS OF THE SINGLE AMINO-ACID SUBSTITUTION OF TYR(82) TO LEU

The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosup pressive properties. One of the amino acids defining the hydrophobic b inding cleft for the macrocycles is Tyr(82), which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structu ral element within the two macrolides. To understand better the influe nce of this amino acid residue in catalytic activity (cis-trans peptid yl prolyl isomerization) and ligand binding properties, a Tyr(82) to L eu site-specific modification of FKBP12 was prepared, purified and cha racterized. Kinetic experiments have demonstrated that the Tyr(82) to Leu modification has a greater effect on catalytic properties than on ligand binding affinities, a result which indicates that these inhibit ors may not be binding as true transition-state analogues. In an addit ional test for cellular function, expression of both wild-type and mut ant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resi stant to rapamycin by deletion of the gene encoding a cytosolic rapamy cin binding protein (RBP1), the yeast homologue of FKBP12, restored wi ld-type drug sensitivity.