Solid-state NMR observation of cysteine and lysine Michael adducts of inactivated estradiol dehydrogenase.
The inactivation of estradiol dehydrogenase by enzyme-generated 3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one is accompanied by the formation of a lysine enaminone. The experiments leading to this conclusion involved degradation of the inactivated enzyme with Pronase and subsequent analysis by solution-state 13C NMR. The present paper reports solid-state 13C NMR experiments on lyophilized intact inactivated enzyme which are free from problems due to Pronase digestion. These experiments combine conventional cross-polarization and magic-angle spinning with selective irradiation of resonances arising from a 13C double label in the steroid. Magnetization transfer between neighboring 13C nuclei is used to simplify the spectra and to identify peaks due to label. The formation of cysteine and lysine Michael adducts of the enzyme is established by comparisons with chemical shifts of solid model adducts.
D015067:17-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors*; D055598:Chemical Phenomena; D002621:Chemistry; D003545:Cysteine; D004789:Enzyme Activation / Q000187:drug effects; D004960:Estradiol Dehydrogenases / Q000037:antagonists & inhibitors*; D004963:Estrenes / Q000494:pharmacology; D005612:Freeze Drying; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D008280:Magnetics; D011402:Pronase; D012632:Secosteroids / Q000494:pharmacology
D004963:Estrenes; D012632:Secosteroids; C056854:3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one; D015067:17-Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases; D011402:Pronase; D008239:Lysine; D003545:Cysteine