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

被引:19
作者
BOSSARD, MJ
BERGSMA, DJ
BRANDT, M
LIVI, GP
ENG, WK
JOHNSON, RK
LEVY, MA
机构
[1] SMITHKLINE BEECHAM PHARMACEUT,DEPT MED CHEM,KING OF PRUSSIA,PA 19406
[2] SMITHKLINE BEECHAM PHARMACEUT,DEPT MOLEC GENET,KING OF PRUSSIA,PA
[3] SMITHKLINE BEECHAM PHARMACEUT,DEPT GENE EXPRESS SCI,KING OF PRUSSIA,PA 19406
[4] SMITHKLINE BEECHAM PHARMACEUT,DEPT BIOMOLEC DISCOVERY,KING OF PRUSSIA,PA 19406
关键词
D O I
10.1042/bj2970365
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosuppressive properties. One of the amino acids defining the hydrophobic binding cleft for the macrocycles is Tyr(82), which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structural element within the two macrolides. To understand better the influence of this amino acid residue in catalytic activity (cis-trans peptidyl prolyl isomerization) and ligand binding properties, a Tyr(82) to Leu site-specific modification of FKBP12 was prepared, purified and characterized. 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 inhibitors may not be binding as true transition-state analogues. In an additional test for cellular function, expression of both wild-type and mutant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resistant to rapamycin by deletion of the gene encoding a cytosolic rapamycin binding protein (RBP1), the yeast homologue of FKBP12, restored wild-type drug sensitivity.
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收藏
页码:365 / 372
页数:8
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