Cytochrome P450 1B1
steroid metabolic process
Increase of human CYP1B1 activities by acidic phospholipids and kinetic deuterium isotope effects on CYP1B1 substrate oxidation.
The effect of phospholipids on the kinetic parameters of three substrates, 7-ethoxy-4 -(trifluoromethyl)coumarin (7-EFC), 7-ethoxycoumarin (7-EC) and 17β-estradiol (E(2)), of human CYP1B1 was studied. In general, anionic phospholipids, phosphatidic acid and cardiolipin increased catalytic efficiency by increasing k(cat) values or decreasing K(m) values. The advantages of using the 7-EFC as a substrate over 7-EC and E(2) include high k(cat), low K(m) and high catalytic efficiency. Spectral binding titrations indicated that the binding affinity of 7-EFC to CYP1B1 in the presence or absence of phospholipids is higher than that of 7-EC or E(2). Furthermore, phosphatidylcholine increased the binding affinity of the substrates to the CYP1B1. High non-competitive intermolecular kinetic deuterium isotope effects (values 5.4-12) were observed for O-deethylation of 7-EFC and 7-EC with deuterium substitution at the ethoxy group, indicating that the C-H bond-breaking step makes a major contribution to the rate of these CYP1B1-catalyzed reactions. However, the intermolecular kinetic deuterium isotope effect is ~2 for the E(2) 4-hydroxylation reaction, indicating that the C-H bond-breaking step contributes only partially to the rate of this CYP1B1-catalyzed reaction. These results indicate that the reaction mechanism of CYP1B1-catalyzed reactions is distinct for each substrate.