Threonine-205 in the F helix of p450 2B1 contributes to androgen 16 beta-hydroxylation activity and mechanism-based inactivation

Four mutants of Thr-205 in cytochrome p450 2B1 were constructed and expressed in Escherichia coli. The Ser-, Ala-, and Val-mutants displayed stable reduced CO difference spectra and were able to metabolize 7-ethoxy-4-(trifluoromethyl)coumarin, testosterone, androstenedione, and benzphetamine. The Arg-mutant displayed an unstable reduced CO difference spectrum at 450 nm, was concomitantly converted to a denatured form with a peak at 422 nm, and showed no catalytic activity with any of the four substrates tested. The Ser-mutant displayed activity and metabolite profiles for testosterone and androstenedione similar to those of the wild-type p450 2B1 (WT). Substitution of Thr-205 with Ala or Val markedly suppressed the 16 beta-hydroxylation activity but exhibited little effect on the 16 alpha-hydroxylation activity for testosterone and androstenedione. Because 16 beta-hydroxylation activity of androgens is a specific p450 2B subfamily marker and residue 205 is located in the F helix, which forms the ceiling of the active site, we postulate that the gamma-hydroxyl side chain of Thr may play an important role in directing the 16 beta-face of testosterone and androstenedione toward the active site. Surprisingly, the Val-mutant retained full activity for benzphetamine demethylation. When mechanism-based inactivators for p450 2B1 were used to evaluate the susceptibility to inactivation, the Val-mutant was resistant to inactivation by 17 alpha-ethynylestradiol and less sensitive to inactivation by 2-ethynylnaphthalene compared with the WT enzyme. Our results demonstrate the importance of Thr-205 in determining substrate specificity and product formation as well as in influencing the susceptibility of p450 2B1 to mechanism-based inactivators.     

Fluoride metabolites after prolonged exposure of volunteers and patients to desflurane.

We examined the metabolism of desflurane in 13 healthy volunteers given 7.35 +/- 0.81 MAC-hours (mean +/- SD) of desflurane and 26 surgical patients given 3.08 +/- 1.84 MAC-hours (mean +/- SD). Markers of desflurane metabolism included fluoride ion measured via an ion-specific electrode, nonvolatile organic fluoride measured after sodium fusion of urine samples, and trifluoroacetic acid determined by a gas chromatographic-mass spectrometric method. In both volunteer and patient groups, postanesthesia serum fluoride ion concentrations did not differ from background fluoride ion concentrations. Similarly, postanesthesia urinary excretion of fluoride ion and organic fluoride in volunteers was comparable to preanesthesia excretion rates. However, small but significant levels of trifluoroacetic acid were found in both serum and urine from volunteers after exposure to desflurane. A peak serum concentration of 0.38 +/- 0.17 mumol/L of trifluoroacetic acid and a peak urinary excretion rate of 0.169 +/- 0.107 mumol/h were detected in volunteers at 24 h after desflurane exposure. Although these increases in trifluoroacetic acid after exposure to desflurane were statistically significant, they are approximately 10-fold less than levels seen after exposure to isoflurane. Thus, desflurane strongly resists biodegradation, but a small amount is metabolized in humans.     

Characterization of halothane oxidation by hepatic microsomes and purified cytochromes P-450 using a gas chromatographic mass spectrometric assay

A sensitive assay for trifluoroacetic acid, the major product of the oxidative metabolism of halothane, has been developed to study the biotransformation of halothane. A selected ion monitoring gas chromatographic mass spectrometric assay measured trifluoroacetic acid levels as low as 1 microM in 100 microliter of reaction mixture. This assay was used to quantitate halothane metabolism in human and rabbit microsomal systems and with purified proteins. Trifluoroacetic acid production was examined as a function of the concentration of substrate present, the amount of microsomal protein used and the length of reaction time. Halothane metabolism in microsomes was linear for at least 30 min, and up to a microsomal protein concentration of 1 mg/ml. In rabbits, phenobarbital and imidazole induced the microsomal metabolism of halothane 7.36- and 18.2-fold, respectively. Imidazole was used because it is a potent inducer of cytochrome P-450 isozyme 3a which is also induced by ethanol. The cytochrome P-450 in microsomes from a single human subject metabolized halothane at a rate comparable to that found in microsomes from phenobarbital- and imidazole-pretreated rabbits. The purified phenobarbital and imidazole inducible cytochromes P-450, isozymes 2 and 3a, catalyzed the oxidation of halothane to trifluoroacetic acid. Cytochrome b5 stimulated the isozyme 3a-catalyzed oxidation of halothane by 19-fold, whereas isozyme 2 catalyzed oxidation was increased 4.3-fold. Antibodies to cytochrome P-450 3a inhibited halothane metabolism by 90% in microsomes from imidazole-pretreated rabbits, suggesting that isozyme 3a catalyzes halothane metabolism in imidazole-pretreated rabbits. In conclusion, the oxidation of halothane to trifluoroacetic acid by cytochrome P-450 isozymes 3a and 2 is enhanced markedly by cytochrome b5.     

I-653 resists degradation in rats

The ability of rats pretreated with phenobarbital to metabolize a new volatile anesthetic, I-653, was compared with the metabolism of halothane, isoflurane, and methoxyflurane. Each anesthetic was administered for 2 hours at 1.6 MAC (inspired). Control rats were given phenobarbital but not exposed to an anesthetic. In rats pretreated with phenobarbital and exposed to I-653, fluoride ion concentrations in serum and excretion of fluoride ion and organic fluoride in the urine were almost indistinguishable from values measured in control rats. In contrast, rats pretreated with phenobarbital metabolized small but significant amounts of isoflurane. In rats pretreated with ethanol and exposed to I-653, the 24-hour excretion of urinary organic fluoride was nearly ten times greater than that observed in control rats. Marked increases in organic fluoride (as high as 1000 times control values) and/or fluoride ion were found in serum and/or urine after anesthesia of phenobarbital-pretreated rats with halothane or methoxyflurane. The relative stability of I-653 indicates that it may possess minimal toxic properties.     

Meperidine carboxylesterase in mouse and human livers

Meperidine carboxylesterase activity was assayed in subcellular fractions of mouse and human liver by coupling the hydrolytic production of ethanol to the reduction of a tetrazolium dye. In mouse liver, the activity was found to be distributed among the mitochondrial, light mitochondrial, and microsomal fractions, whereas in human liver activity was found only in the microsomal fraction. The meperidine carboxylesterases in mouse liver and human liver were inhibited by two irreversible serine hydrolase inactivators (diisopropyl fluorophosphate and paraoxon) and by a reversible transition state analog (trifluoromercaptophenylacetone). Compared to the activities in mouse and human liver microsomes, the activity in mouse liver mitochondria was highly sensitive to the three inhibitors.     

Obligatory role of cytochrome b5 in the microsomal metabolism of methoxyflurane

Cytochrome b5 has recently been shown to be required in the reconstituted cytochrome P-450 system for the metabolism of the volatile anesthetic methoxyflurane [E. Canova-Davis and L. A. Waskell, J. biol. Chem. 259, 2541 (1984)]. To determine whether this observation in the reconstituted system was merely dependent on the particular ratios of the various components or some other fortuitous, unknown factor, or whether cytochrome b5 plays a role in the liver microsomal metabolism of methoxyflurane, the following studies were undertaken. Antibody to rabbit holocytochrome b5 was raised in guinea pigs. The antibody to cytochrome b5 was able to inhibit 75% of the microsomal metabolism of methoxyflurane. This same antibody also inhibited methoxyflurane metabolism in the reconstituted system. When the antibody to cytochrome b5 was treated with purified cytochrome b5 before addition to the microsomes, it did not inhibit methoxyflurane metabolism. Furthermore, the antibody to cytochrome b5 did not inhibit the microsomal metabolism of benzphetamine. This suggests that cytochrome b5 was required for the microsomal metabolism of methoxyflurane. It is possible that cytochrome b5 functioned in the metabolism of methoxyflurane by retaining a specific conformation of cytochrome P-450 and not by transferring the second electron to cytochrome P-450. To explore this possibility, cytochrome b5 was reconstituted with Mn3+-protoporphyrin IX. The Mn3+-protoporphyrin IX derivative retained the conformation of cytochrome b5 but not its electron transfer properties. This manganese derivative of cytochrome b5 was unable to stimulate the metabolism of methoxyflurane. The study demonstrated that cytochrome b5 was obligatory for the microsomal metabolism of methoxyflurane, whereas it was not required for the microsomal N-demethylation of benzphetamine. Moreover, the heme moiety of cytochrome b5 functioned to transfer electrons in this reaction.     

Mutation of tyrosine 190 to alanine eliminates the inactivation of cytochrome P450 2B1 by peroxynitrite

We have previously reported that cytochrome P450 2B1 was inactivated by peroxynitrite and that the decrease in the catalytic activity correlated with an increase in the nitration of tyrosine. Digestion of the peroxynitrite-treated P450 2B1 with Lys C followed by amino acid sequencing of the major nitrotyrosine-containing peptide demonstrated that it spanned residues 160-225. This peptide contains two tyrosine residues at positions 190 and 203. In this study, we mutated Tyr 190 to Ala (Y190A) and Tyr 203 to Ala (Y203A) in wild-type recombinant P450 2B1 (WT) in order to identify the specific residue(s) that is nitrated and to determine whether nitrotyrosine formation is reponsible for the peroxynitrite-mediated inactivation of P450 2B1. All three P450s were expressed in Escherichia coli, purified to homogeneity, and characterized. The catalytic activities for four different substrates of P450 2B1 increased approximately 2-fold for the Y203A mutant, but decreased by about 60% for the Y190A mutant when compared to WT. The addition of peroxynitrite to the P450s resulted in concentration-dependent decreases in the catalytic activities of WT and Y203A, but no loss of the catalytic activities of Y190A. The extent of tyrosine nitration of Y190A by peroxynitrite decreased by approximately 75% as compared with WT or the Y203A protein. Following digestion of the peroxynitrite-modified proteins with Lys C, a major nitrotyrosine-containing peptide was detected from WT and Y203A, but not from Y190A. Collectively, these results indicate that Tyr 190 is the target residue for peroxynitrite-mediated nitration and that nitration of this tyrosine is a responsible for the inactivation of P450 2B1. Modeling studies suggest that Tyr 190 may play a structural role in maintaining the integrity of the protein for maximal activity through hydrogen bonding with Glu 149.