ACTIVITIES OF RECOMBINANT HUMAN CYTOCHROME P450C27 (CYP27) WHICH PRODUCE INTERMEDIATES OF ALTERNATIVE BILE-ACID BIOSYNTHETIC PATHWAYS

The primary physiological significance of cytochrome P450c27 (CYP27) h as been associated with its role in the degradation of the side chain of C-27 steroids in the hepatic bile acid biosynthesis pathway, which begins with 7 alpha-hydroxylation of cholesterol in liver. However, re cognition that in humans P450c27 is a widely or ubiquitously expressed mitochondrial P450, and that there are alternative pathways of bile a cid synthesis which begin with 27-hydroxylation of cholesterol catalyz ed by P450c27, suggests the need to reevaluate the role of this enzyme and its catalytic properties. 27-Hydroxycholesterol was thought to be the only product formed upon reaction of P450c27 with cholesterol. Ho wever, the present study demonstrates that recombinant human P450c27 i s also able to further oxidize 27-hydroxycholesterol giving first an a ldehyde and then 3 beta-hydroxy-5-cholestenoic acid. Kinetic data indi cate that in a reconstituted system, after 27-hydroxycholesterol is fo rmed from cholesterol, it is released from the P450 and then competes with cholesterol for reentry the enzyme active site for further oxidat ion, Under subsaturating substrate concentrations, the efficiencies of oxidation of 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenal t o the acid by human P450c27 are greater than the efficiency of hydroxy lation of cholesterol to 27-hydroxycholesterol indicating that the fir st hydroxylation step in the overall conversion of cholesterol into 3 beta-hydroxy-5-cholestenoic acid is rate-limiting. Interestingly, 3 be ta-hydroxy-5-cholestenoic acid was found to be further metabolized by the recombinant human P450c27, giving two monohydroxylated products wi th the hydroxyl group introduced at different positions on the steroid nucleus.