Cytochrome P450 (P450) 3A4 is the most abundant human P450 and oxidizes a diversity of substrates, including various drugs, steroids, carcinogens, and macrolide natural products. In some reactions, positive cooperativity has been reported in microsomal studies. Flavonoids, e.g., 7,8-benzoflavone (alpha-naphthoflavone, alpha NF), have been shown to stimulate some reactions but not others. In systems containing purified recombinant bacterial P450 3A4, positive cooperativity was seen in oxidations of several substrates, including testosterone, 17 beta-estradiol, amitriptyline, and most notably aflatoxin (AF) B1. With these and other reactions, alpha NF typically reduced cooperativity (i.e., the n value in a Hill plot) while either stimulating or inhibiting reactions. With the substrate AFB1, alpha NF both stimulated 8,9-epoxidation and inhibited 3 alpha-hydroxylation. The same patterns were seen with AFB1 in a fused P450 3A4-NADPH-P450 reductase protein. alpha NF did not alter patterns of activity plotted as a function of NADPH-P450 reductase concentration in systems containing the individual proteins. The patterns of AFB1 oxidation to the two products were modified considerably in systems in which NADPH-P450 reductase was replaced with a flavodoxin or ferredoxin system, iodosylbenzene, or cumene hydroperoxide. AFB2, which differs from AFB1 only in the presence of a saturated 8,9-bond, was not oxidized by P450 3A4 but could inhibit AFB1 oxidation. These and other results are considered in the context of several possible models. The results support a model in which an allosteric site is involved, although the proximity of this putative site to the catalytic site cannot be ascertained as of yet. In order to explain the differential effects of alpha NF and reduction systems on the two oxidations of AFB1, a model is presented in which binding of substrate in a particular conformation can facilitate oxygen activation to enhance catalysis.

Human cytochromes P450 (P450) 1A2 and P450 3A4 were expressed in Escherichia coli, purified, and used in reconstituted oxidation systems. The optimal system for P450 3A4 included a mixture of phospholipids, sodium cholate, cytochrome b5, GSH, and MgCl2. Relatively high catalytic activities were obtained with such a system for aflatoxin (AF) B1 3 alpha-hydroxylation and 8,9-epoxidation. P450 3A4 was more active than P450 1A2 in forming genotoxic AFB1 oxidation products. Analysis of the AFB1 products indicated that P450 3A4 formed AFQ1 and the exo-8,9-epoxide; P450 1A2 formed AFM1, a small amount of AFQ1, and both the exo- and endo-8,9-epoxides. The endo epoxide is essentially nongenotoxic in the umu test, as found previously in bacterial mutagenicity assays [Iyer, R. S., Coles, B. F., Raney, K. D., Thier, R., Guengerich, F. P., and Harris, T. M. (1994) J. Am. Chem. Soc. 116, 1603-1609]. 7,8-Benzoflavone (alpha-naphthoflavone, alpha NF) stimulated AFB1 (exo) 8,9-epoxidation and inhibited 3 alpha-hydroxylation in human liver microsomes and a reconstituted P450 3A4 system but was a potent inhibitor of all reactions catalyzed by P450 1A2. Plots of AFB1 3 alpha-hydroxylation and 8,9-epoxidation vs AFB1 concentration were sigmoidal in both human liver microsomes and the reconstituted P450 3A4 system. The results are consistent with the view that P450 3A4 is a major human liver P450 enzyme involved in AFB1 activation, although the in vivo situation may be more complex due to the presence of the enzyme in the gastrointestinal tract.

The oxidation of benzo[a]pyrene (B[a]P) was examined using reconstituted systems prepared with recombinant human cytochrome P450 (P450) enzymes 1A1, 1A2, 2C8, 2C10, 2E1, and 3A4 and with microsomes prepared from Saccharomyces cerevisiae expressing recombinant human P450s 2C8, 2C9, and 2C18. Products measured by HPLC included the 3- and 9-phenols, the 4,5-, 7,8-, and 9,10-dihydrodiols (detected in the presence of epoxide hydrolase), and products in the polar fraction eluting immediately after the void volume. The most active enzyme in all reactions was P450 1A1. P450 3A4 and P450 1A2 formed appreciable amounts of several of the products, including the 3-phenol. P450 2C enzymes and P450 2E1 formed relatively low amounts of all B[a]P products. Consideration of these patterns along with knowledge of levels of expression of the P450s in human tissues and previous results with microsomes leads to the conclusion that P450 1A1 should dominate the oxidation of B[a]P in tissues where it is present and inducible. In human liver the level of P450 1A1 is low and P450 3A4, P450 2C subfamily enzymes, and P450 1A2 probably all contribute. Of the human P450s considered here, P450 1A2 was the most active hepatic enzyme forming the 7,8-dihydrodiol. 7,8-Benzoflavone stimulated the oxidation of B[a]P by P450 3A4 and inhibited the oxidations catalyzed by P450 1A2. The extent of inhibition of P450 1A1 was less (than with P450 1A2), probably due to the rapid oxidation of 7,8-benzoflavone by P450 1A1. The major 7,8-benzoflavone product appears to be the 5,6-oxide.

Many catalytic activities of cytochrome P450 (P450)3A4 More than 40 P4501 enzymes are found in a single mammalian species (2). The proteins constitute a superfamily and collectively contribute extensively to the oxidation of xenobiotic chemicals (e.g. drugs, carcinogens, pesticides, alkaloids, and other natural products) and also endobiotics (e.g. steroids, eicosanoids, fat-soluble vitamins, fatty acids) (3-6). The contributions of these P450 enzymes to metabolism in humans are well recognized, particularly regarding issues of drug clearance (7-9). There is general agreement that, in most humans, P450 3A4 is the most abundant of the P450s in both liver and small intestine (8, 9); it can constitute up to 60% of the total P450 in the liver (10). The intestinal enzyme has been implicated in variation in the bioavailability of many orally administered drugs (11). P450 3A4 has a very broad range of substrates, with more than 60 drugs having been already identified (9). These vary widely in structure, and one of the questions about this enzyme has been the molecular basis of its broad catalytic specificity (12, 13). Other mechanistic questions involve the basis of the sigmoidal plots of enzyme velocity versus substrate seen with some compounds (14 -16) and the stimulation of activity by chemicals other than the substrate (14,17,18). The purified enzyme, along with other P450 3A subfamily enzymes, is much more sensitive to its reconstitution environment than are most other P450s (19, 20). A variety of components have been reported to stimulate catalytic activity, including long chain unsaturated phosphatidylcholines (21), phosphatidylserine (20,22), ionic detergents (21, 22), GSH (23), divalent cations (24, 25), and b 5 (19,21). Not all of these components are directly relevant to the membrane-bound enzyme, but Mg 2ϩ has been shown to stimulate activity of the enzyme in microsomes (24, 25) and antibodies raised against b 5 can inhibit some catalytic activities of P450 3A4 in microsomes (19,25). Somewhat surprisingly, certain catalytic activities of P450 3A4 are quite refractory to alterations in lipids and b 5 (24, 26).In order to better understand this complex but important system, we initiated a systematic investigation of some of the system components on individual steps in the catalytic cycle of purified recombinant P450 3A4 (14,19,20,(23)(24)(25). A general conclusion about the role of b 5 in modulating P450 reactions has been that electron transfer from b 5 to P450 occurs in step 4 of Scheme 1 (27). Recently we found qualitative evidence that b 5 could also stimulate the reduction of P450 3A4 by the flavoprotein NADPH-P450 reductase (24). We now report that apo-b 5 (devoid of heme) can replace b 5 in the efficient oxidation of the prototypic P450 substrates testosterone and nifedipine and that apo-b 5 can also replace b 5 in the facilitating electron flow from NADPH-P450 reductase to P450 3A4, in the absence of electron transfer from b 5 or modulation of the E m,7 of P450 3A4.

Recombinant cytochrome P450 (P450) 3A4 was most active in nifedipine and testosterone oxidation in a system including NADPH-P450 reductase, cytochrome b5 (b5), a semisynthetic phospholipid mixture plus cholate, glutathione, and MgCl2. The MgCl2 effect could be seen with high concentrations of Ca2+ or Sr2+ but not readily when these cations were replaced with monovalent cations. The divalent cation effect was also seen in liver microsomes. Part of the basis of this effect appears to be enhanced rates of b5 reduction, as judged from studies on deletions of reconstitution components and analysis of steady-state spectral studies. Rapid reduction of ferric P450 3A4 to ferrous was dependent upon the presence of substrate, either testosterone or ethylmorphine. When testosterone was present, reduction was also highly dependent upon the presence of b5 and Mg2+. In the case of the substrate ethylmorphine, the need to add b5 and Mg2+ to obtain optimal reduction rates was less pronounced. These patterns are consistent with the dramatic dependence of testosterone 6 beta-hydroxylation on b5 and the lack of dependence of ethylmorphine N-demethylation on b5. Our interpretation is that divalent cations stimulate electron transfer from NADPH-P450 reductase to several acceptors and that substrates and b5 can bind to P450 3A4 to influence its rate of reduction by the reductase. P450 3A4 catalyzed testosterone 6 beta-hydroxylation within Escherichia coli cells. The reactions could be supported by E. coli cytosol or by purified E. coli flavodoxin and NADPH-flavodoxin reductase. Spinach ferredoxin and NADPH-ferredoxin reductase also supported catalytic activities.(ABSTRACT TRUNCATED AT 250 WORDS)