ENZYMATIC CHARACTERIZATION OF A NOVEL BOVINE LIVER DIHYDRODIOL DEHYDROGENASE - REACTION-MECHANISM AND BILE-ACID DEHYDROGENASE-ACTIVITY

Bovine liver cytosolic dihydrodiol dehydrogenase (DD3) has been charac terized by its unique dihydrodiol dehydrogenase activity for trans-ben zenedihydrodiol (trans-1,2-dihydrobenzene-1,2-diol) with the highest a ffinity and the greatest velocity among three multiple forms of dihydr odiol dehydrogenases (DD1-DD3). It is the first time that DD3 has show n a significant dehydrogenase activity for (S)-(+)-1-indanol with low K-m value (0.33 +/- 0.022 mM) and high K-cat value (25 +/- 0.79 min(-1 )). The investigation of the product inhibition of (S)-(+)-1-indanol w ith NADP(+) versus 1-indanone and NADPH clearly showed that the enzyma tic reaction of DD3 may follow a typical ordered Bi Bi mechanism simil ar to many aldo/keto reductases. Additionally, DD3 was shown to cataly ze the dehydrogenation of bile acids (lithocholic acid, taurolithochol ic acid anti taurochenodeoxycholic acid) having no 12-hydroxy groups w ith low K-m values (17 +/- 0.65, 33 +/- 1.9 and 890 +/- 73 mu M, respe ctively). In contrast, DD1, 3 alpha-hydroxysteroid dehydrogenase, show s a broad substrate specificity for many bile acids with higher affini ty than those of DD3. Competitive inhibition of DD3 with androsterone against dehydrogenase activity for (S)-(+)-1-indanol, trans-benzenedih ydrodiol or lithocholic acid suggests that these three substrates bind to the same substrate binding site of DD3, different from the case of human liver bile acid binder/dihydrodiol dehydrogenase (Takikawa, H., Stolz, A., Sugiyama, Y., Yoshida, H., Yamamoto, M. and Kaplowitz, N. (1990) J. Biol. Chem. 265, 2132-2136). Considering the reaction mechan ism, DD3 may also play an important role in bile acids metabolism as w ell as the detoxication of aromatic hydrocarbons.