Aldehyde oxidase activity and inhibition in hepatocytes and cytosolic fractions from mouse, rat, monkey and human.

Aldehyde oxidase (AO) is a cytosolic enzyme that contributes to the Phase I metabolism of xenobiotics in human and preclinical species. We compared AO activity in cytosol and cryopreserved hepatocytes from human, monkey, rat and mouse livers to assess species differences. We also evaluated possible species differences in drug interactions using seven drugs known to inhibit human cytosolic AO i.e. raloxifene, perphenazine, menadione, maprotiline, ketoconazole, erythromycin, and estradiol. AO activity was measured using the formation of vanillic acid from vanillin. The rate of vanillic acid formation was 2 +/- 0.2 nmol/min/mg in human liver cytosol and 0.79 +/- 0.45 nmol/min/million cells in cryopreserved human hepatocytes. AO activity (V(max,app)) was highest in monkey and lowest in rat. Mouse liver cytosol had the lowest K(m,app) (1.44 +/- 0.16 microM) and highest intrinsic clearance (8.97 ml/min/mg) and rat liver cytosol the highest K(m,app) (10.9 +/- 1.2 microM) and lowest intrinsic clearance (0.47 ml/min/mg). There was a 4.25-fold difference in AO activity between the 5 human hepatocyte preparations. Drug interaction studies with the seven marketed drugs revealed marked species-specific inhibition. Our data indicates major differences in the rate of AO metabolism, and inhibition of AO across species, indicating that results from animal studies cannot be safely extrapolated to humans. Cryopreserved hepatocytes and cytosolic fractions from animals and humans provide qualitatively similar data within the species.

Topological changes in the CYP3A4 active site probed with phenyldiazene: effect of interaction with NADPH-cytochrome P450 reductase and cytochrome b5 and of site-directed mutagenesis.

The active site topology of heterologously expressed CYP3A4 purified from an Escherichia coli expression system was examined using phenyldiazene. Incubation of CYP3A4 with phenyldiazene and subsequent oxidation yielded all four potential N-phenylprotoporphyrin IX regioisomers derived from attack on an available nitrogen atom in pyrrole rings B, A, C, or D (N(B):N(A):N(C):N(D) = 6:73:7: 13). Further study using 28 active site mutants showed that substitution of residues closer to the heme, Ala-305, Thr-309, or Ala-370, with a larger residue caused the most drastic changes in regioisomer formation, which reflected the location of each amino acid residue replaced in a CYP3A4 homology model. Previous studies have suggested a conformational change in CYP3A4 upon binding of NADPH-cytochrome P450 reductase (CPR) or cytochrome b(5) (b(5)). Therefore, regioisomer formation was also compared in the absence of redox partners and in the presence of CPR, b(5), or both. Formation of all four regioisomers in CYP3A4 wild type, particularly the minor ones, was reduced in the presence of b(5). CPR also greatly decreased the three minor isomers but increased the major isomer significantly. The presence of b(5) and CPR restored minor isomer formation and suppressed the enhancement of N(A) formation caused by CPR alone. Interestingly, the effects of the redox partners differed among representative active site mutants. In particular, the increase in N(C) upon substitution of Ala-370 with Phe was significantly reversed in the presence of redox partners, strongly suggesting that a conformational change occurs around pyrrole ring C due to protein-protein interactions between CYP3A4 and CPR or b(5).

Homotropic versus heterotopic cooperativity of cytochrome P450eryF: a substrate oxidation and spectral titration study.

P450eryF is the only bacterial P450 to show cooperativity of substrate binding and oxidation. However, the studies reported so far have provided evidence only for homotropic cooperativity of P450eryF but not for heterotropic cooperativity. Therefore, oxidation of 7-benzyloxyquinoline (7-BQ) and 1-pyrenebutanol (1-PB) by P450eryF A245T and spectral binding of 9-aminophenanthrene (9-AP) to wild-type P450eryF were investigated in the presence of various effectors. The addition of steroids and flavones caused no stimulation but rather moderate inhibition of 7-BQ or 1-PB oxidation by P450eryF A245T. However, the binding affinity of 9-AP was significantly increased in the presence of androstenedione or alpha-naphthoflavone (ANF). A comparative study with CYP3A4 revealed a similar increase in the binding affinity of 9-AP for the enzyme at low ANF concentrations but some competition at higher ANF concentrations. These studies, to our knowledge, provide the first report of heterotropic cooperativity in P450eryF as well as spectroscopic evidence for simultaneous presence of two ligand molecules in the CYP3A4 active site.

Differential oxidation of mifepristone by cytochromes P450 3A4 and 3A5: selective inactivation of P450 3A4.

The principal enzyme involved in the oxidation of mifepristone is cytochrome P450 3A4 (CYP3A4), which undergoes mechanism-based inactivation by the drug. However, no information is available on the interaction with CYP3A5, the second most abundant CYP3A enzyme in adult human liver. Oxidation of mifepristone by recombinant CYP3A4 produced mono- and didemethylated products and one C-hydroxylated metabolite, as reported previously. However, CYP3A5 produced only the demethylated metabolites. The apparent V(max) and K(M) values for formation of the monodemethylated product by CYP3A4 and CYP3A5 were 46 and 30 nmol/min/nmol P450, and 36 and 16 microM, respectively. Unlike CYP3A4, CYP3A5 was not inactivated by mifepristone. The basis of this differential susceptibility was explored using site-directed mutants in which a CYP3A4 residue was converted to its 3A5 counterpart. Surprisingly, none of these replacements caused a significant decrease in CYP3A4 inactivation by mifepristone. Examination of selected CYP3A4 mutants at 20 other positions indicated that the relative formation rate of the C-hydroxylated product could not account for the differential susceptibility of CYP3A4 and 3A5. Together these results indicate that mifepristone fails to orient itself in the CYP3A5 active site in such a way that its propylenic group is accessible for oxidation, thus rendering CYP3A5 unable to produce the C-hydroxylated product or putative ketene that leads to enzyme inactivation. Identification of mifepristone as a selective mechanism-based inactivation of CYP3A4 may be very useful in distinguishing between the two major CYP3A enzymes collectively responsible for the oxidative metabolism of over half of the drugs currently in use.

7-Benzyloxyquinoline oxidation by P450eryF A245T: finding of a new fluorescent substrate probe.

The main objective of the present study was to find a fluorescent substrate probe for cytochrome P450eryF (P450eryF). P450eryF is a bacterial P450 that catalyzes the hydroxylation of 6-deoxyerythronolide B at the 6S position, a necessary step in the biosynthesis of erythromycin. The lack of a conserved threonine residue in the I-helix, in contrast to other P450s, makes P450eryF unable to oxidize other substrates. A recent study [Xiang et al. (2000) J. Biol. Chem. 275, 35999-36006] has shown that the substitution of Ala-245 by threonine confers on P450eryF significant testosterone hydroxylase activity. Therefore, we investigated various known fluorescent P450 substrates with P450eryF wild-type as well as two mutants, A245S and A245T. Among the various fluorescent compounds tested, 7-benzyloxyquinoline (7-BQ) was found to be the most suitable probe for P450eryF A245T, with rates of oxidation being lower for A245S and wild-type enzyme. The steady-state kinetics of 7-BQ oxidation by A245T are sigmoidal (V(max) = 0.71 nmol/min/nmol, n = 2.18, and S(50) = 132 microM). alpha-Naphthoflavone (alpha-NF), a well-known activator of CYP3A4, did not stimulate 7-BQ oxidation by A245T, although the S(50) value for alpha-NF binding to wild-type P450eryF was similar to P450 3A4. Interestingly, spectral binding studies of wild-type P450eryF and A245T with ketoconazole and miconazole showed differential binding behaviors. Titration of wild-type with ketoconazole and miconazole and of A245T with miconazole showed the expected type-II binding. However, titration of A245T with ketoconazole produced a spectrum similar to type-I. Inhibition studies showed that both ketoconazole and miconazole are able to inhibit 7-BQ oxidation by A245T, although miconazole showed a slightly higher potency. In brief, the present study reports the discovery of 7-BQ as the first fluorescent and only the second unnatural substrate, and of miconazole as an effective P450eryF inhibitor.

Site-directed mutagenesis of cytochrome P450eryF: implications for substrate oxidation, cooperativity, and topology of the active site.

The role of five active-site residues (Phe-78, Gly-91, Ser-171, Ile-174, and Leu-175) has been investigated in P450eryF, the only bacterial P450 known to show cooperativity. The residues were selected based on two-ligand-bound P450eryF structures and previous mutagenesis studies of other cytochromes P450. To better understand the role of these residues in substrate catalysis and cooperativity, each mutant was generated in the wild-type and A245T background, a substitution that enables P450eryF to oxidize testosterone and 7-benzyloxyquinoline (7-BQ). Replacement of Phe-78 with tryptophan decreased cooperativity of 9-aminophenanthrene binding, with little effect on testosterone binding or oxidation. Interestingly, substitution of Gly-91 with alanine or phenylalanine abolished the type-I spectral change elicited by testosterone and significantly decreased testosterone hydroxylation. However, G91A/A245T showed a 4-fold higher k(cat) value with 7-BQ compared with A245T. Replacement of Ser-171 with alanine or phenylalanine did not alter cooperativity of testosterone binding but significantly decreased binding affinity and oxidation of testosterone and 7-BQ. The only mutant that exhibited an increased testosterone binding affinity and increased rates of testosterone and 7-BQ oxidation was I174F. Substitution of Ile-175 with phenylalanine decreased testosterone and 7-BQ oxidation. Reaction with phenyldiazene showed that P450eryF may be much more open above pyrrole ring B than other cytochromes P450 and indicated significant changes in active-site topology in some of the mutants. The study suggests a crucial role of residues Ser-171, Ile-174, and Leu-175, which are part of a distal ligand site, in addition to the proximal Gly-91 in determining the oxidative properties of P450eryF.

Midazolam oxidation by cytochrome P450 3A4 and active-site mutants: an evaluation of multiple binding sites and of the metabolic pathway that leads to enzyme inactivation.

Midazolam (MDZ) oxidation by recombinant CYP3A4 purified from Escherichia coli and 30 mutants generated at 15 different substrate recognition site positions has been studied to determine the role of individual residues in regioselectivity and to investigate the possible existence of multiple binding sites. Initial results showed that oxidation of MDZ by CYP3A4 causes time- and concentration-dependent enzyme inactivation with K(I) and k(inact) values of 5.8 microM and 0.15 min(-1), respectively. The different time courses of MDZ hydroxylation by mutants that predominantly formed 1'-OH MDZ as opposed to 4-OH MDZ provided strong evidence that the 1'-OH MDZ pathway leads to CYP3A4 inactivation. Correlational analysis of 1'-OH formation versus 4-OH formation by the mutants supports the inference that the two metabolites result from the binding of MDZ at two separate sites. Thus, substitution of residues Phe-108, Ile-120, Ile-301, Phe-304, and Thr-309 with a larger amino acid caused an increase in the ratio of 1'-OH/4-OH MDZ formation, whereas substitution of residues Ser-119, Ile-120, Leu-210, Phe-304, Ala-305, Tyr-307, and Thr-309 with a smaller amino acid decreased this ratio. Kinetic analyses of nine key mutants revealed that the alteration in regioselectivity is caused by a change in kinetic parameters (V(max) and K(M)) for the formation of both metabolites in most cases. The study revealed the role of various active-site residues in the regioselectivity of MDZ oxidation, identified the metabolic pathway that leads to enzyme inactivation, and provided an indication that the two proposed MDZ binding sites in CYP3A4 may be partially overlapping.