Inactivation of Escherichia coli-expressed rabbit cytochrome P-450 2C enzymes by 17 beta-substituted steroids.
The specific inactivation of rabbit cytochromes P-450 2C by 17 beta-substituted steroids has been investigated by using purified, Escherichia coli-expressed enzymes. The expressed P-450s provided a means to characterize accurately the effects of 21,21-dichloroprogesterone, 21,21-dichloropregnenolone, 21-chloro-21-fluoropregnenolone, pregn-5,20-diene-3 beta-ol and pregn-4,20-diene-3-one on progesterone hydroxylation by P-450 2C5, 2C4, 2C3 and 2C3v. Previous studies using rabbit liver microsomes had suggested that 21-chloro-21-fluoropregnenolone is a selective inactivator of 2C5, a progesterone 21-hydroxylase. Studies of the expressed P-450 2C forms showed little selectivity of 21,21-dichloroprogesterone, pregn-5,20-diene-3 beta-ol or pregn-4,20-diene-3-one, whereas 21,21-dichloropregnenolone and 21-chloro-21-fluoropregnenolone preferentially inactivate 2C5. The data indicate the importance of progesterone 21-hydroxylase activity in facilitating selective mechanism-based inactivation of 2C subfamily P-450s by 21,21-dihalogenated steroids. Studies of the inactivation of P-450 2C16, a progesterone 16 alpha-hydroxylase, by the three dihalogenated steroids yielded results consistent with previous findings of 16 alpha-hydroxylase inactivation in rabbit liver microsomes from the inbred B/J strain. Additionally, two mutants, 2C3v:V113A and 2C3v:V113A, T364N were created which confer progesterone 21-hydroxylation on 2C3v. The single mutant, a 6 beta- and 21-hydroxylase, is inactivated rapidly by all three of the 21,21-dihalogenated steroids, whereas the double mutant, a 16 alpha- and 21-hydroxylase, is preferentially inactivated by 21,21-dichloroprogesterone.(ABSTRACT TRUNCATED AT 250 WORDS)
Klekotka
P A
PA
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, USA.
Richardson
T H
TH
Johnson
E F
EF
Halpert
J R
JR
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D003001:Cloning, Molecular; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004926:Escherichia coli; D007700:Kinetics; D016296:Mutagenesis; D011284:Pregnenolone / Q000031:analogs & derivatives / Q000494:pharmacology*; D011374:Progesterone / Q000031:analogs & derivatives / Q000494:pharmacology*; D011817:Rabbits; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors* / Q000235:genetics
D065607:Cytochrome P-450 Enzyme Inhibitors; D011374:Progesterone; D011284:Pregnenolone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase