Theoretical kinetics of sequential metabolism in vitro. Study of the formation of 16 alpha-hydroxyandrostenedione from testosterone by purified rat P450 2C11.
P450 2C11 from rat liver is known to metabolize testosterone to 2 alpha-, 16 alpha-, and 6 beta-hydroxytestosterone, and to androstenedione and 16 alpha-hydroxyandrostenedione. Because Waxman (J. Biol. Chem. 259, 15481-15490) has reported that the enzyme converts androstenedione to 16 alpha-hydroxyandrostenedione, it seemed likely that the metabolite was formed from testosterone by way of androstenedione. Indeed, we have found that P450 2C11 does not convert 16 alpha-hydroxytestosterone to 16 alpha-hydroxyandrostenedione to any significant extent and, therefore, that the metabolite is formed from testosterone almost solely by way of androstenedione. To determine whether some of the 16 alpha-hydroxyandrostenedione might be formed directly from the androstenedione-enzyme complex, we developed an approach by which it is possible to calculate the amount of the androstenedione, released into the medium, relative to the amount of the androstenedione-enzyme complex that is converted directly to 16 alpha-hydroxyandrostenedione under initial conditions when the concentration of released androstenedione will be negligible. The approach uses two factors: factor A is the androstenedione/(androstenedione + 16 alpha-hydroxyandrostenedione) present at the end of the incubation, and factor B corrects for the amount of released androstenedione that recombines with the enzyme and is converted to 16 alpha-hydroxyandrostenedione. Although the values of both factors A and B will vary with the concentrations of testosterone and preformed androstenedione present in the incubation mixtures and with the duration of incubation, the value of A*B will be independent of these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)
Sugiyama
K
K
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Nagata
K
K
Gillette
J R
JR
Darbyshire
J F
JF
D000735:Androstenedione / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002250:Carbon Radioisotopes; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D006915:Hydroxytestosterones / Q000378:metabolism; D007700:Kinetics; D008432:Mathematical Computing; D051381:Rats; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000302:isolation & purification / Q000378:metabolism*; D013739:Testosterone / Q000378:metabolism*
D002250:Carbon Radioisotopes; D006915:Hydroxytestosterones; D013739:Testosterone; D000735:Androstenedione; C018202:16-hydroxyandrost-4-en-3,17-dione; C045261:16-hydroxytestosterone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase