Abstract:
Two enzyme mechanisms were examined: the 21-dehydroxylation
of corticosteroids by the anaerobe Eubacterium l en tum, and the
hydroxylation of steroids by fungal cytochrome P450.
Deuterium labelling techniques were used to study the enzymic
dehydroxylation. Corticosteroids doubly labelled (2H) at the C-21
position were incubated with a culture of Eubacterium lentum. It
was found that t he enzymic dehydroxylation proceeded with the
loss of one 2H f rom C-21 per molecule of substrate. The kinetic
isotope ef fect f or the reaction was found to be k~kD = 2. 28.
These results suggest that enzyme/substr ate binding in this case
may proceed via t he enol form of the substrate. Also , it appears
that this binding is, at least in part, the rate determining
step of t he reaction.
The hydroxylation of steroids by fungal cytochrome P450 was
examined by means of a product study. Steroids with a double bond
at the A8 (9), ~( lO ), or ~ (ll) position were synthesized. These
steroids were then incubated with fungal strains known to use a
cytochrome P450 monooxygenase to hydroxylate at positions allylic
to these doubl e bonds. The products formed in these incubations
indicated that the double bonds had migrated during allylic
hydroxylat ion. This suggests that a carbon centred radical or ion
may be an intermediate i n the cytochrome P450 cat alytic cycle.
