STRUCTURAL-ANALYSIS OF THE INTERACTION BETWEEN THE SIDE-CHAIN OF SUBSTRATES AND THE ACTIVE-SITE OF LANOSTEROL 14-ALPHA-DEMETHYLASE (P-450(14DM)) OF YEAST

The role of the side-chain of lanosterol in the enzyme-substrate interaction of yeast P-450(14DM) (lanosterol 14-alpha-demethylase) was analyzed with lanosterol derivatives having functional groups on the side-chain. Purified P-450(14DM) from Saccharomyces cerevisiae catalyzed 14-alpha-demethylation of 26-hydroxylanosterol and 25-hydroxy-24,25-dihydrolanosterol with a lower activity than lanosterol and 24,25-dihydrolanosterol. This enzyme demethylated the (Z)-24-ethylidene-24,25-dihydrolanosterol with a low rate, but did not metabolize the E-isomer. The apparent K(m) of 26-hydroxylanosterol was 10.8-mu-M, which was higher than that of lanosterol, but lower than that of 24,25-dihydrolanosterol. On the other hand, competition experiments suggested that the affinity of 25-hydroxy-24,25-dihydrolanosterol and (Z)-24-ethylidene-24,25-dihydrolanosterol for P-450(14DM) was significantly lower than that of 24,25-dihydrolanosterol. Integration of the present results with the preceding ones (Aoyama, Y., Yoshida, Y., Sonoda, Y. and Sato, Y. (1991) Biochim. Biophys. Acta, 1081, 262-266 and Aoyama, Y. and Yoshida, Y. (1991) Biochem. Biophys. Res. Commun., 178, 1064-1071) suggests that yeast P-450(14DM) recognizes two parts of the side-chain, the structure around C-24 and the terminal fork consisting of C-25, C-26 and C-27.