title: Covalent attachment of diethylstilbestrol to glutamate dehydrogenase: implications for allosteric regulation.
abstract: An affinity labeling reagent for the estrogenic-binding site of bovine liver L-glutamate dehydrogenase (EC 1.4.1.3) was prepared by conversion of diethylstilbestrol to its alkylating analogue, bromoacetyldiethylstilbestrol. Under standard assay conditions, the analogue acted as a reversible allosteric ligand with regulatory activity much like that of diethylstilbestrol. However, incubation of the enzyme with the alkylating agent in the presence of DPNH resulted in a permanent decrease in glutamate (X form) and an increase in alanine (Y form) activities, and in covalent attachment of diethylstilbestrol in the ratio of 1 mol per subunit (of particle weight 52,000). The brominated analogue behaved as an affinity label that mimicked the allosteric effects of diethylstilbestrol. Diethylstilbestrol protection of the enzyme against alkylation by bromoacetylated sterol suggested competition for the same binding site, while ADP protection indicated a shift of protein equilibrium into the X form. The diethylstilbestrol-enzyme compound was desensitized (relative to the native enzyme) to allosteric reagents such as ADP and GTP. The results were consistent with conformational freezing of the modified protein molecule into the Y form.
mesh_terms: D000227:Adenine Nucleotides; D000477:Alkylating Agents; D000818:Animals; D001665:Binding Sites; D001669:Biochemical Phenomena; D001671:Biochemistry; D002417:Cattle; D004054:Diethylstilbestrol; D005246:Feedback; D005615:Freezing; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D006150:Guanine Nucleotides; D006898:Hydroxylamines; D008099:Liver / Q000201:enzymology; D009243:NAD; D014316:Tritium
chemical_list: D000227:Adenine Nucleotides; D000477:Alkylating Agents; D006150:Guanine Nucleotides; D006898:Hydroxylamines; D009243:NAD; D014316:Tritium; D004054:Diethylstilbestrol; D005969:Glutamate Dehydrogenase