Deuterium isotope effects on A-ring and D-ring metabolism of testosterone by CYP2C11: evidence for dissociation of activated enzyme-substrate complexes.
Cytochrome P450 systems are unusual in that many of them can convert a substrate to a number of different metabolites by several possible kinetic mechanisms. Steady-state equations describing the deuterium isotope effects for mechanisms in which different orientations of the substrate relative to the perferryl oxygen in the active site of the enzyme are achieved before a hydrogen (or possibly an electron) is abstracted have been derived and solved (Gillette et al., 1994). These equations have been used to elucidate the kinetic mechanisms by which CYP2C11 converts testosterone to 2 alpha-hydroxytestosterone on the one hand and 16 alpha-hydroxytestosterone and androstenedione on the other. We have synthesized testosterone-2,2,4,6,6-2H5 and compared its metabolism by CYP2C11 with that of nondeuterated testosterone. In this system, deuterated 2 alpha-hydroxytestosterone would be formed by a deuterium abstraction pathway via the active oxygen intermediate (EOSw) and the D-ring metabolites would be formed by non-deuterium abstraction pathways from active oxygen intermediates represented by (EOSx). The results revealed that testosterone in the activated enzyme-substrate complexes, (EOSw) and (EOSx), does not change orientations while it is in the active site of CYP2C11. Instead, two of the noncompetitive experiments indicated that testosterone is able to dissociate from the (EOS) complexes and reassociate in either the same or different orientations. A third noncompetitive experiment suggested that testosterone in the (EOS) complexes does not change orientations while it is in the active site of CYP2C11, nor does it dissociate from the (EOS) complexes; instead, the pattern of metabolite formation is governed almost solely by the orientation of testosterone in the (ESw) and ESx) complexes.
Darbyshire
J F
JF
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Gillette
J R
JR
Nagata
K
K
Sugiyama
K
K
D001189:Aryl Hydrocarbon Hydroxylases; D001667:Binding, Competitive; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003903:Deuterium; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008968:Molecular Conformation; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013739:Testosterone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase