The reaction of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide with an essential carboxy group of human placental estradiol 17 beta-dehydrogenase.
Estradiol 17 beta-dehydrogenase from human placenta was inactivated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), a specific reagent for modification of carboxyl group in a protein. Inactivation of the dehydrogenase by EDC progressed in time-dependent, concentration-dependent and ionic strength-dependent manners. In the presence of NAD+ or NADP+, the inactivation rate by EDC was markedly accelerated. NMN+ and acetylpyridine-DPN+ also appeared to increase the rate of modification of the dehydrogenase by EDC, but no acceleration of the inactivation was observed in the presence of the reduced forms of those nucleotides. Inactivation by EDC in the presence of NAD+ was accompanied by incorporation of 2 mol of NAD+/mol of dimer (Mr = 68,000) of the enzyme. When the dehydrogenase was incubated with EDC together with estriol, the activity was lost slower than that of control without the steroid. Similarly, the inactivation was prevented by addition of estrone and estradiol as well as aminoestrogens. From those results, we suggest that the carboxyl group essential for the function of estradiol 17 beta-dehydrogenase is buried in the enzyme molecule, and the positive charge of nicotinamide N1 of the oxidized form of the cofactor plays an important role for enhancement of the inactivation by EDC.
D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D002234:Carbodiimides; D002264:Carboxylic Acids; D004960:Estradiol Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004964:Estriol / Q000378:metabolism; D004970:Estrone / Q000378:metabolism; D005022:Ethyldimethylaminopropyl Carbodiimide; D005260:Female; D006801:Humans; D009243:NAD / Q000378:metabolism; D009994:Osmolar Concentration; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013329:Structure-Activity Relationship
D002234:Carbodiimides; D002264:Carboxylic Acids; D009243:NAD; D004970:Estrone; D015067:17-Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases; D004964:Estriol; D005022:Ethyldimethylaminopropyl Carbodiimide