Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g
Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6..beta..-hydroxyprogesterone and 16..cap alpha..-hydroxyprogesterone), two secondary metabolites (6..beta..,16..cap alpha..-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16..cap alpha..-hydroxyprogesterone). The K/sub m,app/ for the formation of these products from progesterone was determined to be approximately 0.5 ..mu..M, while the K/sub m,app/ for metabolism of 6..beta..- and 16..cap alpha..-hydroxyprogesterone was found to be 5-10 ..mu..M. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 ..mu..M, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6..beta..,16..cap alpha..-dihydroxyprogesterone and 6-keto-16..cap alpha..-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6..beta..-hydroxyprogesterone or 16..cap alpha..-hydroxyprogesterone. Incubation of 6..beta..-hydroxyprogesterone with a reconstituted system in an atmosphere of /sup 18/I/sub 2/ resulted in > 90% incorporation of /sup 18/O in the 16..cap alpha..-position of 6..beta..,16..cap alpha..-dihydroxyprogesterone but no incorporation of /sup 18/O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O/sub 2/, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.
- Research Organization:
- Roche Institute of Molecular Biology, Nutley, NJ (USA)
- OSTI ID:
- 7188294
- Journal Information:
- Biochemistry; (United States), Vol. 27:15
- Country of Publication:
- United States
- Language:
- English
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PROGESTERONE
BIOCHEMICAL REACTION KINETICS
DERIVATIZATION
LABELLING
OXIDATION
CARBON 14 COMPOUNDS
CYTOCHROMES
GAS CHROMATOGRAPHY
ISOENZYMES
LIVER
MASS SPECTROSCOPY
METABOLITES
OXYGEN 18
RATS
ANIMALS
BODY
CHEMICAL REACTIONS
CHROMATOGRAPHY
DIGESTIVE SYSTEM
EVEN-EVEN NUCLEI
GLANDS
HORMONES
ISOTOPES
KETONES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MAMMALS
NUCLEI
ORGANIC COMPOUNDS
ORGANS
OXYGEN ISOTOPES
PIGMENTS
PREGNANES
PROTEINS
REACTION KINETICS
RODENTS
SEPARATION PROCESSES
SPECTROSCOPY
STABLE ISOTOPES
STEROID HORMONES
STEROIDS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques