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 Tyr82, 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 Tyr82 to Leu site-specific modification of FKBP12 was prepared, purified and characterized. Kinetic experiments have demonstrated that the Tyr82 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 (RPB1), the yeast homologue of FKBP12, restored wild-type drug sensitivity.
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January 1994
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Research Article|
January 15 1994
Catalytic and ligand binding properties of the FK506 binding protein FKBP12: effects of the single amino acid substitution of Tyr82 to Leu Available to Purchase
M J Bossard;
M J Bossard
*Department of Medical Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
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D J Bergsma;
D J Bergsma
†Departments of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, U.S.A.
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M Brandt;
M Brandt
*Department of Medical Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
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G P Livi;
G P Livi
‡Gene Expression Sciences SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, U.S.A.
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W K Eng;
W K Eng
§Biomolecular Discovery, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, U.S.A.
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R K Johnson;
R K Johnson
*Department of Medical Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
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M A Levy
M A Levy
*Department of Medical Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1994 The Biochemical Society, London
1994
Biochem J (1994) 297 (2): 365–372.
Citation
M J Bossard, D J Bergsma, M Brandt, G P Livi, W K Eng, R K Johnson, M A Levy; Catalytic and ligand binding properties of the FK506 binding protein FKBP12: effects of the single amino acid substitution of Tyr82 to Leu. Biochem J 15 January 1994; 297 (2): 365–372. doi: https://doi.org/10.1042/bj2970365
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