Partial purification and kinetic properties of a soluble estrogen glucuronyltransferase from pig intestine.

A soluble enzyme from the small intestine of the pig, capable of conjugating uridine diphosphate glucuronic acid with 17/3-estradiol, has been purified 5to IO-fold. The glucuronide formed was identified as 17P-estradiol J-monoglucuronide by paper chromatography and microchemical reactions. This estrogen glucuronyltransferase is present in the 150,000 x g supernatant and shows an absolute requirement for UDP-glucuronic acid. The glucuronidation reaction is optimal in Tris-HCl buffer between pH 8.0 and 8.6. Magnesium ions and cysteine increase the activity of the enzyme; p-chloromercuribenzoate and iodoacetamide are inhibitors of the enzyme. Km values were found to be 0.74 X 10m6 M for 17/3-estradiol and 0.84 x 10m4 M for UDP-glucuronic acid. The glucuronyltransferase is stable for at least 3 months at -20°, while it loses 50% of its initial activity within 20 days when kept at +5”. The enzyme shows a temperature optimum of 42’. The reaction has an activation energy of 15 kcal per mole within the range of 22’ to 42’. ATP competes with UDP-glucuronic acid for the enzyme, whereas UDP inhibits the enzyme noncompetitively. The following steroidal and nonsteroidal substrates are not attacked to any significant extent by the estrogen glucuronyltransferase: estriol, l’/cr-estradiol, dehydroepiandrosterone, testosterone, bilirubin, and p-nitrophenol. However, e&one is conjugated by the enzyme and also acts as a competitive inhibitor of the glucuronidation of 17P-estradiol; the inhibition constant, Rip for estrone was calculated to be 0.79 x 10V6 M. The similarity of the Ki value for estrone and the K, value for 17/3-estradiol makes it likely that only one enzyme catalyzes the glucuronidation of both 17P-estradiol and estrone.