Structural analysis of the interaction between the side-chain of substrates and the active site of lanosterol 14 alpha-demethylase (P-450(14)DM) of yeast.
The role of the side-chain of lanosterol in the enzyme-substrate interaction of yeast P-450(14)DM (lanosterol 14 alpha-demethylase) was analyzed with lanosterol derivatives having functional groups on the side-chain. Purified P-450(14)DM 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 Km of 26-hydroxylanosterol was 10.8 microM, 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(14)DM 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(14)DM 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.
Aoyama
Y
Y
Faculty of Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan.
Yoshida
Y
Y
Sonoda
Y
Y
Sato
Y
Y
D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D007700:Kinetics; D007810:Lanosterol / Q000031:analogs & derivatives / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D058886:Sterol 14-Demethylase; D013379:Substrate Specificity
D007810:Lanosterol; C015316:lanostenol; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase