The general considerations underlying active-site-directed irreversible enzyme inhibition (affinity labeling of enzymes) have been applied to the study of steroid-binding proteins, restricting attention to cytoplasmic androgen and progestagen receptors. There are two requisites for the affinity labeling of a steroid receptor. The steroid must bind reversibly at the active site of the receptor and possess a functional group capable of forming a covalent bond with an amino acid residue either within (endo) or adjacent to (exo) the binding site. The first requisite seems relatively easy to fulfill. The progesterone receptor of chick oviduct forms a highly stable (kd ∼ 10-10) complex with progesterone and competition studies show that other steroids form complexes somewhat less stable but with kd ∼ 10-10. Thus, in the absence of progesterone, the progesterone-binding protein should strongly bind other steroids. The second requisite is more difficult. The amino acids in the polypeptide chain at the binding site are not known. Cysteine may be one because it has been shown for both a rat prostate 5α-dihydrotestosterone receptor and the chick oviduct progesterone receptor that sulfhydryl groups are involved either in the binding of the steroid to the receptor or in the maintenance of the active structure of the protein. It is assumed that one or both of the oxygen functions of 5α-dihydrotestosterone (C-3 and C-17) and of progesterone (C-3 and C-20) is involved in binding. A reactive substituent at a position near one of these oxygen functions would probably be closer to the polypeptide chain in the steroid–receptor complex than a more remotely positioned substituent and consequently would be more likely to undergo reaction. Four general classes of chemical reactions that are used in attempts at affinity labeling apply here such as alkylation reactions, photochemical insertion reactions, disulfide bond formation, and mercaptide bond formation.
All Science Journal Classification (ASJC) codes
- Molecular Biology