Cytochrome P450 7A1 Cholesterol 7α-Hydroxylation

Abstract

Cytochromes P450 (P450) 2 enzymes are hemoproteins and most commonly catalyze oxidation reactions ( Fig. 1) (3,4). In mammals, P450 enzymes play important roles in the metabolism of both endogenous substrates (e.g. fatty acids, steroids, eicosanoids) and exogenous substrates (e.g. drugs, carcinogens, pesticides). Most mammalian P450 enzymes, especially the "drug-metabolizing" P450 enzymes (e.g. P450s 1A2, 2C9, 2D6), show relatively low catalytic activity (k cat Ͻ 10 min Ϫ1 , often ϳ1 min Ϫ1 ) (3-5) compared with some of the classical microbial P450 enzymes with high activities, e.g. P450s 101A1, 102A1 (k cat Ͼ 10 3 min Ϫ1 ) (5). On the other hand, a few mammalian P450 enzymes, e.g. rat P450 2B1 and 4A1, rabbit P450 4A7, and human P450 7A1, have been reported to have higher catalytic activities (k cat Ն 50 min Ϫ1 ) (6 -9). Among these latter mammalian P450s, P450 7A1 has been reported to have quite high catalytic activity toward the substrate cholesterol (as high as 5.8 s Ϫ1 ) (9). This enzyme is well known as the cholesterol 7␣-hydroxylase, the first and ratelimiting enzyme in the classic pathway of bile acid synthesis, with a daily elimination of 400 -600 mg of cholesterol in humans (10, 11). A k cat /K m value reported for recombinant P450 7A1 was 4 ϫ 10 5 M Ϫ1 s Ϫ1, although the cholesterol 7␣-hydroxylation activity in human liver microsomes has been reported to be Ͻ0.13 pmol/s/mg of microsomal protein (k cat ϳ 0.06 s Ϫ1 ) (12)(13)(14). This efficiency (k cat /K m ) of cholesterol 7␣-hydroxylation by recombinant P450 7A1 is ϳ10-fold higher than that of testosterone 6␤-hydroxylation by P450 3A4, which is one of the faster reactions catalyzed by the drug-metabolizing P450 enzymes (15). However, to our knowledge no detailed kinetic studies on cholesterol 7␣-hydroxylation activity by P450 7A1 are available, and why P450 7A1 shows such high catalytic activity is unknown.We have been interested in the reaction cycles of several mammalian P450s with low catalytic efficiencies, including the rate-limiting steps in reactions (15-18). Our previous reports on the reactions of P450s 1A2, 2A6, 2D6, and 3A4 show that the C-H bond-breaking step of each reaction is mainly or partially rate-limiting, as judged by kinetic deuterium isotope effects. Kinetic analysis on P450 7A1, with its high catalytic efficiency, was done for comparison with low efficiency P450 reactions to better understand differences between them and to define why P450 rates vary so much.In the present study we analyzed individual reaction steps and developed a detailed kinetic model of P450 7A1 reaction cycle with individual rate constants. The substrate binding, C-H bond-breaking, and product release steps are not ratelimiting. The results indicate that the first electron transfer step, although as rapid as observed with other mammalian P450s, becomes rate-limiting in the P450 7A1 reaction. EXPERIMENTAL PROCEDURESChemicals-Cholesterol, 17␣-ethynylestradiol, dansyl chloride, 4,4-dimethylaminopyridine, protocatechuate, protocatechuate dioxygenase, HP␤CD, an...

Conversion of 7-Dehydrocholesterol to 7-Ketocholesterol Is Catalyzed by Human Cytochrome P450 7A1 and Occurs by Direct Oxidation without an Epoxide Intermediate

“…The product was defined by co-migration with an authentic standard and an identical fragmentation pattern, as elaborated further under studies with P450 7A1 (see below). The measured rate was 0.014 Ϯ 0.002 pmol/min/mg of protein, which is 100-fold less than that of cholesterol 7␣-hydroxylation in human liver microsomes (2.5 Ϯ 1.6 pmol/ min/mg of protein) (40). The estimated rate of 7-dehydrocholesterol oxidation in liver microsomes may be compromised by the competition of the endogenous cholesterol, in that the measured K m for (free) cholesterol for P450 7A1 is ϳ1 M (40) and an IC 50 of 1 M (for 7-ketocholesterol) has been reported for cholesterol 7␣-hydroxylation (20).…”

“…Enzyme Preparations-Recombinant P450 7A1 with a C-terminal His 6 tag was expressed in Escherichia coli using a plasmid originally provided by I. Pikuleva (Case Western Reserve University, Cleveland, OH) and purified as described elsewhere (40). Recombinant P450 3A4 with a C-terminal His 5 tag (41) was expressed in E. coli and purified as described elsewhere (42).…”

“…(40). For P450 3A4 reactions, a "5ϫ P450 protein premix" was prepared with slight modification of a previously described method (45).…”

“…Measurement of [25,26,26,26,27,27,27-d 7 ]-7-Dehydrocholesterol Oxidation in Human Liver Microsomes-The amount of cholesterol in a pool of 10 human liver microsomal samples (seven males and three females) was first quantified as described (40). Aliquots of the pool of the 10 human liver microsomal samples containing exactly 50 nmol of cholesterol were added to a 1.0-ml reaction mixture containing 100 nmol of [25,26,26,26,27,27,27-d 7 ]-7-dehydrocholesterol and 3.1 mM HP␤CD in 50 mM potassium phosphate buffer (pH 7.4).…”

Inhibition of Human Cytochrome P450 3A4 by Cholesterol

“…Microsomes-The amount of cholesterol in a set of 10 pooled human liver microsomes (seven males and three females) (31) was first quantified as described previously (33). Aliquots of 10 pooled human liver microsomal samples, including exactly 25 nmol of cholesterol, were added to 1.0 ml of reaction mixture containing different amounts of cholesterol (0 -225 nmol) and 3.1 mM HP␤CD in 50 mM potassium phosphate buffer (pH 7.4).…”